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 brown dog tick Rhipicephalus sanguineus sensu lato (s.l.) is a species complex comprising three main mitochondrial lineages, namely tropical, temperate and southeast European lineages. Despite its medical and veterinary importance, little attention has been paid to the genetic lineage of this species in Southeast Asia. Rhipicephalus sanguineus s.l. from Malaysia was investigated genetically, for the first time, using the mitochondria-encoded cytochrome c oxidase subunit I (COI) and 16S ribosomal RNA (16S) genes. Specifically, a pair of primers was developed to amplify the COI sequences in the present study. Both genes unambiguously assigned Malaysian material into the tropical lineage of R. sanguineus s.l. The 16S sequences were highly conserved; no variation site was observed. The COI sequences revealed slightly higher variation by recovering four haplotypes, one of which is restricted to the northernmost of Peninsular Malaysia. This finding will be a stepping stone in promoting more biological studies of this species complex in this region.
Anaplasmosis and ehrlichiosis are of serious health concern worldwide for animals and humans. In the present study, we report the occurrence of Anaplasma platys and Ehrlichia canis in dogs and Rhipicephalus sanguineus sensu lato (s.l.) ticks from Peninsular Malaysia using a nested polymerase chain reaction assay based on amplification of the 16S rRNA gene. Anaplasma platys was detected from dogs and ticks with prevalence rates of 3.3% (8/240) and 2.9% (4/140), respectively. On the other hand, 12.9% (31/240) of the dogs and 0.7% (1/140) of the ticks were tested positive for E. canis. Additionally, co-infections of A. platys and E. canis with Babesia or Hepatozoon protozoa were also noted in this study. Double infection (E. canis + B. gibsoni) was observed in tick, whereas triple infections (E. canis + A. platys + B. vogeli and E. canis + A. platys + H. canis) were found in dogs. This study represents the first evidence of A. platys DNA in R. sanguineus s.l. in Malaysia.
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.
Mites are known causes of allergic diseases. Currently, identification of mites based on morphology is difficult if only one mite is isolated from a (dust) sample, or when only one gender is found, or when the specimen is not intact especially with the loss of the legs. The purpose of this study was to use polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) of the ITS2 gene, to complement the morphological data for the identification of mites to the species level. For this, six species were cultured: Dermatophagoides pteronyssinus, D. farinae, Blomia tropicalis, Tyrophagus putrescentiae, Aleuroglyphus ovatus and Glycycometus malaysiensis. Genomic DNA of the mites was extracted, quantified, amplified and digested individually with restriction enzymes. Hinf I and Ple I differentiated the restriction patterns of D. pteronyssinus and D. farinae. Bfa I and Alu I enzymes differentiated B. tropicalis and G. malaysiensis. Ple I enzyme was useful for the differentiation between T. putrescentiae and A. ovatus. Bfa I was useful for the differentiation of G. malaysiensis from the rest of the species. In conclusion, different species of mites can be differentiated using PCR-RFLP of ITS2 region. With the established PCR-RFLP method in this study, identification of these mites to the species level is possible even if complete and intact adult specimens of both sexes are not available. As no study to date has reported PCR-RFLP method for the identification of domestic mites, the established method should be validated for the identification of other species of mites that were not included in this study.
The genetic identity of Ixodes granulatus ticks was determined for the first time in Taiwan. The phylogenetic relationships were analyzed by comparing the sequences of mitochondrial 16S ribosomal DNA gene obtained from 19 strains of ticks representing seven species of Ixodes and two outgroup species (Rhipicephalus sanguineus and Haemaphysalis inermis). Four major clades could be easily distinguished by neighbour-joining analysis and were congruent by maximum-parsimony method. All these I. granulatus ticks of Taiwan were genetically affiliated to a monophyletic group with highly homogeneous sequences (92.2-99.3% similarity), and can be discriminated from other Ixodes species and other genera of ticks with a sequence divergence ranging from 11.7 to 30.8%. Moreover, intraspecific analysis revealed that two distinct lineages are evident between the same species of I. granulatus ticks collected from Taiwan and Malaysia. Our results demonstrate that all these I. granulatus ticks of Taiwan represent a unique lineage distinct from the common vector ticks (I. ricinus complex) for Borrelia burgdorferi spirochetes.
Varroa jacobsoni was first described as a natural ectoparasitic mite of the Eastern honeybee (Apis cerana) throughout Asia. It later switched host to the Western honeybee (A. mellifera) and has now become a serious pest of that bee worldwide. The studies reported here on genotypic, phenotypic and reproductive variation among V. jacobsoni infesting A. cerana throughout Asia demonstrate that V. jacobsoni is a complex of at least two different species. In a new classification V. jacobsoni is here redefined as encompassing nine haplotypes (mites with distinct mtDNA CO-I gene sequences) that infest A. cerana in the Malaysia Indonesia region. Included is a Java haplotype, specimens of which were used to first describe V. jacobsoni at the beginning of this century. A new name, V. destructor n. sp., is given to six haplotypes that infest A. cerana on mainland Asia. Adult females of V. destructor are significantly larger and less spherical in shape than females of V. jacobsoni and they are also reproductively isolated from females of V. jacobsoni. The taxonomic positions of a further three unique haplotypes that infest A. cerana in the Philippines is uncertain and requires further study. Other studies reported here also show that only two of the 18 different haplotypes concealed within the complex of mites infesting A. cerana have become pests of A. mellifera worldwide. Both belong to V. destructor, and they are not V. jacobsoni. The most common is a Korea haplotype, so-called because it was also found parasitizing A. cerana in South Korea. It was identified on A. mellifera in Europe, the Middle East, Africa, Asia, and the Americas. Less common is a Japan/Thailand haplotype, so-called because it was also found parasitizing A. cerana in Japan and Thailand. It was identified on A. mellifera in Japan, Thailand and the Americas. Our results imply that the findings of past research on V. jacobsoni are applicable mostly to V. destructor. Our results will also influence quarantine protocols for bee mites, and may present new strategies for mite control.
The phoretic association between Macrocheles muscaedomesticae and flies that inhabited poultry manure in a poultry farm in Sungai Buloh, Selangor, Malaysia was studied. The effects of temperature, relative humidity and fly abundance on phoretic rates also were investigated. The most abundant fly species found was Musca domestica; Musca sorbens, Chrysomyia megacephala and Ophyra chalcogaster were present in relatively large numbers. Representatives of ten families of mites were found on collected Mu. domestica. The most common mite was Ma. muscaedomesticae (Macrochelidae), found on all four species of flies mentioned above. The highest infestation (2.0%) occurred on O. chalcogaster but Mu. domestica had the highest average number infested (5.7). The ventral part of the housefly's abdomen was the most common site of mite attachment. Usually only one mite was found attached per fly. The highest phoretic rate recorded was 64.4 Ma. muscaedomesticae per 1000 Mu. domestica. There was no correlation between phoretic rates and Ma. muscaedomesticae abundance, nor was relative humidity a factor. However, a positive correlation was recorded in this host species between phoretic rates and temperature.
Pulau Tioman is a popular tourist island off the east coast of Peninsula Malaysia, known for its vast areas of intact vegetation. Six tick species have been recorded on the island but little is known about their local ecology or public health importance. To facilitate studies of the tick fauna of Pulau Tioman, a checklist of all species and host associations is presented with the first host records, from Pulau Tioman, of Amblyomma helvolum from the Asian water monitor (Varanus salvator) and Amblyomma varanense from the keeled rat snake (Ptyas carinata). A pictorial key is also presented for all tick species known from the island.
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.
Acari community structure and function associated with delayed pig carrion decomposition has not been examined. In this study, 18 swine carcasses were studied in central Texas, USA, during two consecutive summers (2013 and 2014). Samples of ca. 400 g soil were collected from beneath, aside, and 5 m away from each pig carcass over 180 days. Mites from soil samples were extracted using Berlese funnels and identified to order and family levels and classified according to ecological function. In total 1565 and 1740 mites were identified from the 2013 and 2014 soil samples, respectively. Significant differences were determined for mite community structure at order and family levels temporally on carrion (e.g., day 0 × day 14) regardless of treatments and between soil regions where mites were collected (e.g., soil beneath vs. soil 5 m away from carrion). However, no significant differences were found in mite community structure at the order level between pig carrion with and without delayed Diptera colonization (i.e., treatments). Analysis at the family level determined a significant difference across treatments for both summers. Ecological function of mites did not change significantly following the delayed decomposition of pig carcasses. However, detritivores and fungivores were significant indicator groups during the pig carrion decomposition process. Furthermore, 13 phoretic mite species associated with eight forensically important beetle species were documented. Data from this study indicated that the rate of nutrient flow into the soil impacted associated arthropod communities; however, detecting such shifts depends on the taxonomic resolution being applied.
Ticks attaching to ear canals of humans and animals are the cause of otoacariasis, common in rural areas of Nepal. The plant Clerodendrum viscosum is used in multiple indigenous systems of medicine by ethnic communities in the Indo-Nepali-Malaysian region. Visiting the Chitwan National Park, we learned that in indigenous medicine, flower extract of C. viscosum is utilized to treat digestive disorders and extracts from leaves as tick repellent to prevent ticks from invading or to remove them from the ear canal. The objective of our study was to provide support to indigenous medicine by characterizing the in vivo effect of leave extracts on ticks under laboratory conditions and its phytochemical composition. We collected plant parts of C. viscosum (leaves and flowers) and mango (Mangifera indica) leaves at the Chitwan National Park, previously associated with repellent activity to characterize their effect on Ixodes ricinus ticks by in vivo bioassays. A Q-ToF high-resolution analysis (HPLC-ESI-QToF) was conducted to elucidate phenolic compounds with potential repellent activity. Clerodendrum viscosum and M. indica leaf extracts had the highest tick repellent efficacy (%E = 80-100%) with significant differences when compared to C. viscosum flowers extracts (%E = 20-60%) and phosphate-buffered saline. Phytochemicals with tick repellent function as caffeic acid, fumaric acid and p-coumaric acid glucoside were identified in C. viscosum leaf extracts by HPLC-ESI-QToF, but not in non-repellent flower extracts. These results support the Nepali indigenous medicine application of C. viscosum leaf extracts to repel ticks. Additional research is needed for the development of natural and green repellent formulations to reduce the risks associated with ticks resistant to acaricides.
The prevalence of house dust mite (HDM) allergy, especially in Asian countries with rapid urbanization, has been increasing. House dust mites thrive in places with relatively high humidity. With the combination of climate change, naturally high humidity, and urbanization, tropical countries like Malaysia are becoming a hotspot for HDM allergy fast. With a previously reported sensitization rate of between 60 and 80%, it is a worrying trend for Malaysia. However, due to incomplete and out-of-date data, as seen by the limited study coverage in the past, these numbers do not paint a complete picture of the true HDM allergy scene in Malaysia. This review briefly discusses the HDM fauna, the HDM sensitization rate, the common diagnosis and therapeutic tools for HDM allergy in Malaysia, and makes suggestions for possible improvements in the future. This review also highlights the need of more comprehensive population-based prevalence studies to be done in Malaysia, encompassing the three main HDMs-Dermatophagoides pteronyssinus, Dermatophagoides farinae, and Blomia tropicalis-as the lack of up-to-date studies failed to give a clearer picture on the current scenario of HDM allergy in Malaysia. Future studies will be beneficial to the nation in preparing a better blueprint for the management and treatment of HDM allergy.
Due to the role of Ixodes ricinus (L.) (Acari: Ixodidae) in the transmission of many serious pathogens, personal protection against bites of this tick is essential. In the present study the essential oils from 11 aromatic Egyptian plants were isolated and their repellent activity against I. ricinus nymphs was evaluated Three oils (i.e. Conyza dioscoridis L., Artemisia herba-alba Asso and Calendula officinalis L.) elicited high repellent activity in vitro of 94, 84.2 and 82%, respectively. The most active essential oil (C. dioscoridis) was applied in the field at a concentration of 6.5 µg/cm2 and elicited a significant repellent activity against I. ricinus nymphs by 61.1%. The most repellent plants C. dioscoridis, C. officinalis and A. herba-alba yielded essential oils by 0.17, 0.11 and 0.14%, respectively. These oils were further investigated using gas chromatography-mass spectrometry analysis. α-Cadinol (10.7%) and hexadecanoic acid (10.5%) were the major components of C. dioscoridis whereas in C. officinalis, α-cadinol (21.2%) and carvone (18.2%) were major components. Artemisia herba-alba contained piperitone (26.5%), ethyl cinnamate (9.5%), camphor (7.7%) and hexadecanoic acid (6.9%). Essential oils of these three plants have a potential to be used for personal protection against tick bites.