Research Article |
Corresponding author: Ernieenor Faraliana Che Lah ( erniee@imr.gov.my ) Academic editor: Dmitry Apanaskevich
© 2015 Ernieenor Faraliana Che Lah, Salmah Yaakop, Mariana Ahamad, Shukor Md Nor.
This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Citation:
Che Lah EF, Yaakop S, Ahamad M, Md Nor S (2015) Molecular identification of blood meal sources of ticks (Acari, Ixodidae) using cytochrome b gene as a genetic marker. ZooKeys 478: 27-43. https://doi.org/10.3897/zookeys.478.8037
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Blood meal analysis (BMA) from ticks allows for the identification of natural hosts of ticks (Acari: Ixodidae). The aim of this study is to identify the blood meal sources of field collected on-host ticks using PCR analysis. DNA of four genera of ticks was isolated and their cytochrome b (Cyt b) gene was amplified to identify host blood meals. A phylogenetic tree was constructed based on data of Cyt b sequences using Neighbor Joining (NJ) and Maximum Parsimony (MP) analysis using MEGA 5.05 for the clustering of hosts of tick species. Twenty out of 27 samples showed maximum similarity (99%) with GenBank sequences through a Basic Local Alignment Search Tool (BLAST) while 7 samples only showed a similarity range of between 91–98%. The phylogenetic trees showed that the blood meal samples were derived from small rodents (Leopoldamys sabanus, Rattus tiomanicus and Sundamys muelleri), shrews (Tupaia glis) and mammals (Tapirus indicus and Prionailurus bengalensis), supported by 82–88% bootstrap values. In this study, Cyt b gene as a molecular target produced reliable results and was very significant for the effective identification of ticks’ blood meal. The assay can be used as a tool for identifying unknown blood meals of field collected on-host ticks.
Ticks, Blood meal, Vector control, hosts, Cytochrome b
The tick is a member of the class Arachnida that belongs to the sub-class Acari. It relies heavily on other animals as hosts to complete their life cycle (
Research on the identification of a natural host for ticks is valuable and is the main goal of a blood meal analysis (BMA). The analysis is a tool used to identify the hosts of blood feeding arthropods (
The progressive development of molecular analysis has resulted in the use of markers that were originally designed to study phylogenetic relationships among vertebrates being applied to determine the blood meal origin of haematophagous arthropods (
Analysis of blood meals of local ticks to determine their natural hosts is still poorly understood. There is a critical necessity to document information of potential hosts for local ticks as part of the nation’s preparedness for emerging and re-emerging infections. Thus, the aim of this study was to identify the blood meal sources of ticks in Peninsular Malaysia based on the cytochrome b (Cyt b) gene. The obtained Cyt b genes were then used to construct a phylogenetic tree for the clustering of hosts of tick species.
On-host ticks were collected from four different states of Peninsular Malaysia (Figure
The species of animals were identified by their morphological traits following
Prior to DNA extraction, each engorged tick was individually washed 3 times with sterile distilled water. Extraction of DNA using QIAamp Mini Kit (Qiagen, Germany) was performed according to the manufacturer’s protocols. DNA of ticks was extracted by adding 80 µl of PBS buffer and 100 µl of ATL buffer into the sample. The ticks were then macerated using sterile tips for 5 minutes before adding of 20 µl of proteinase K. The samples were incubated at 56 °C (6 hours) for complete lyses. The following steps were the same as those in the manufacture’s protocols. The DNA was then used for subsequent PCR.
A portion of mitochondrial DNA, Cyt b gene was amplified by Polymerase Chain Reaction (PCR) with a set of vertebrate-universal primers and reaction conditions as described by
The PCR product was excised with a sterile gel cutter and purified using 5 Prime PCR Agarose Gel Extract Mini Kit (Hamburg, Germany) according to the manufacturer’s protocols. The purified product was then sent to the sequencing service company, Medigene Sdn. Bhd. in Petaling Jaya, Selangor. The sequencing was bi-directional for all specimens and the primer combination for this step was the same as that used in the PCR amplification. Sequencing results were exported as FASTA sequence files. The Cyt b gene sequences of samples were aligned using ClustalW multiple alignment of BioEdit (
The obtained sequences were then compared with available sequences in the GenBank database using the Basic Local Alignment Search Tool search (NCBI website, http://www.ncbi.nlm.nih.gov/BLAST/) for the identification of the host species. This approach was reported to be simple and robust for rapid comparison of query sequences to database sequences leading to species identification (
The clustering analysis for all sequences of hosts was carried out by performing phylogenetic analysis using MEGA software (version 5.05). For distance analysis, a neighbor-joining (NJ) tree was generated from a Kimura two-parameter distance matrix. Maximum-parsimony (MP) analysis was performed with Tree-Bisection-Reconnection (TBR) heuristic algorithm to reconstruct a character based phylogenetic tree. Internal branches of both trees were statistically supported by bootstrapping with 1,000 replications. In this study, Apodemus sylvaticus (GenBank Accession no. AJ298599.1) was selected as an outgroup for Cyt b gene.
A total of 27 engorged ticks collected from four different localities (Table
Code samples | Tick species | Locality | Ecology |
K1a | Amblyomma testudinarium | Krau Wildlife Reserved, Pahang | Pristine tropical rainforest |
K2a | Dermacentor sp. | Krau Wildlife Reserved, Pahang | Pristine tropical rainforest |
K3c | Dermacentor sp. | Krau Wildlife Reserved, Pahang | Pristine tropical rainforest |
K4b | Amblyomma sp. | Krau Wildlife Reserved, Pahang | Pristine tropical rainforest |
K5b | Amblyomma sp. | Krau Wildlife Reserved, Pahang | Pristine tropical rainforest |
SBN 01 | Ixodes granulatus | Labu, Negeri Sembilan | Scrubs |
SBN12_1 | Dermacentor sp. | Labu, Negeri Sembilan | Scrubs |
SBN23_1 | Ixodes granulatus | Labu, Negeri Sembilan | Scrubs |
SBN 14 | Ixodes sp. | Labu, Negeri Sembilan | Scrubs |
JBB01_1 | Dermacentor sp. | Janda Baik, Pahang | Riverine vegetation |
JBB03_1 | Haemaphysalis sp. | Janda Baik, Pahang | Riverine vegetation |
JBB03_2 | Haemaphysalis sp. | Janda Baik, Pahang | Riverine vegetation |
JBB03_3 | Haemaphysalis sp. | Janda Baik, Pahang | Riverine vegetation |
JBB03_4 | Haemaphysalis sp. | Janda Baik, Pahang | Riverine vegetation |
HL01 | Ixodes granulatus | Hulu Langat, Selangor | Pristine tropical rainforest |
HL02 | Ixodes granulatus | Hulu Langat, Selangor | Pristine tropical rainforest |
HL03_2 | Ixodes granulatus | Hulu Langat, Selangor | Pristine tropical rainforest |
HL03 | Ixodes granulatus | Hulu Langat, Selangor | Pristine tropical rainforest |
HL04_15 | Ixodes granulatus | Hulu Langat, Selangor | Pristine tropical rainforest |
HL02_4 | Ixodes granulatus | Hulu Langat, Selangor | Pristine tropical rainforest |
HL02_5 | Ixodes granulatus | Hulu Langat, Selangor | Pristine tropical rainforest |
HL02_3 | Ixodes granulatus | Hulu Langat, Selangor | Pristine tropical rainforest |
HL07 | Ixodes granulatus | Hulu Langat, Selangor | Pristine tropical rainforest |
HL02_1 | Ixodes granulatus | Hulu Langat, Selangor | Pristine tropical rainforest |
GT23_2 | Ixodes granulatus | Gunung Tebu, Terengganu | Secondary growth |
GT23_3 | Ixodes granulatus | Gunung Tebu, Terengganu | Secondary growth |
GT23_5 | Ixodes granulatus | Gunung Tebu, Terengganu | Secondary growth |
Vertebrate DNA was successfully amplified from 27 engorged ticks. The amplification of a single fragment encoding a 623 bp sequence of the Cyt b gene yielded the expected amplification products (Figure
Amplification of Cyt b gene produced 623 bp of PCR products from ticks species. Lane 1: unfed ticks (negative control); Lane 2: vertebrate DNA (positive control); Lanes 3–8: DNA of field-collected ticks (Ixodes sp., Dermacentor sp., Amblyomma sp., Amblyomma testudinarium, Haemaphysalis sp., Haemaphysalis sp.,) and Lanes M1, M2: 100 bp DNA ladder (Bioron, Germany).
Code samples | Tick species | Host species (morphological) | % similarity with GenBank (species) |
K1a | Amblyomma testudinarium | Tapirus indicus | 99 (Tapirus indicus) |
K2a | Dermacentor sp. | Tapirus indicus | 99 (Tapirus indicus) |
K3c | Dermacentor sp. | Tapirus indicus | 99 (Tapirus indicus) |
K4b | Amblyomma sp. | Tapirus indicus | 99 (Tapirus indicus) |
K5b | Amblyomma sp. | Tapirus indicus | 99 (Tapirus indicus) |
SBN 01 | Ixodes granulatus | Rattus tiomanicus | 99 (Rattus tiomanicus) |
SBN12_1 | Dermacentor sp | Rattus tiomanicus | 99 (Rattus tiomanicus) |
SBN23_1 | Ixodes granulatus | Rattus tiomanicus | 98 (Rattus tiomanicus) |
SBN 14 | Ixodes sp. | Rattus tiomanicus | 99 (Prionailurus bengalensis) |
JBB01_1 | Dermacentor sp. | Tupaia glis | 99 (Tupaia glis) |
JBB03_1 | Haemaphysalis sp. | Sundamys muelleri | 91 (Sundamys muelleri) |
JBB03_2 | Haemaphysalis sp. | Sundamys muelleri | 91 (Sundamys muelleri) |
JBB03_3 | Haemaphysalis sp. | Sundamys muelleri | 91 (Sundamys muelleri) |
JBB03_4 | Haemaphysalis sp. | Sundamys muelleri | 91 (Sundamys muelleri) |
HL01 | Ixodes granulatus | Leopoldamys sabanus | 99 (Leopoldamys sabanus) |
HL02 | Ixodes granulatus | Leopoldamys sabanus | 99 (Leopoldamys sabanus) |
HL03_2 | Ixodes granulatus | Leopoldamys sabanus | 99 (Leopoldamys sabanus) |
HL03 | Ixodes granulatus | Leopoldamys sabanus | 97 (Leopoldamys sabanus) |
HL04_15 | Ixodes granulatus | Sundamys muelleri | 98 (Sundamys muelleri) |
HL02_4 | Ixodes granulatus | Leopoldamys sabanus | 99 (Leopoldamys sabanus) |
HL02_5 | Ixodes granulatus | Leopoldamys sabanus | 99 (Leopoldamys sabanus) |
HL02_3 | Ixodes granulatus | Leopoldamys sabanus | 99 (Leopoldamys sabanus) |
HL07 | Ixodes granulatus | Leopoldamys sabanus | 99 (Leopoldamys sabanus) |
HL02_1 | Ixodes granulatus | Leopoldamys sabanus | 99 (Leopoldamys sabanus) |
GT23_2 | Ixodes granulatus | Leopoldamys sabanus | 99 (Leopoldamys sabanus) |
GT23_3 | Ixodes granulatus | Leopoldamys sabanus | 99 (Leopoldamys sabanus) |
GT23_5 | Ixodes granulatus | Leopoldamys sabanus | 99 (Leopoldamys sabanus) |
From the 27 aligned DNA sequences, a total of 575-bp portion of the Cyt b gene was used for analysis. Out of 575 characters from Cyt b fragments, 252 variable sites were detected, among which 203 (35.3%) variable characters were parsimony-informative while 49 (8.5%) characters were parsimony-uninformative. Additionally, the conserved sites were constituted by 323 (56.1%) characters showing that Cyt b gene is a very conserved gene in the mtDNA.
The mitochondrial Cyt b gene sequences of host species were grouped into 2 major clades by NJ and MP analysis; one group of small rodents and another consisting of shrews and large mammals. The tree topology showed that all 27 host sequences examined fell into two distinct genetic lineages: Clade A (consists of L. sabanus, R. tiomanicus and S. muelleri) and Clade B (consists of T. glis, P. bengalensis and T. indicus). NJ tree topology revealed a distinction with 95% bootstrap value for Clade A but a lower bootstrap value of 85% for Clade B (Figure
Neighbor-joining tree constructed from 28 sequences (including one outgroup sequence) of the Cyt b gene. The numbers at the branches stand for bootstrap values 70% and above of 1000 replications. Genera of ticks represented by blue for Ixodes sp., orange for Dermacentor sp., violet for Haemaphysalis sp. and green for Amblyomma sp.
Seven parsimonious trees were produced by the MP analysis using equally weighted TBR. The best tree had 497 steps (Figure
Maximum parsimony tree constructed from 28 sequences (including one outgroup sequence) of the Cyt b gene. The numbers at the branches stand for bootstrap values 70% and above of 1000 replications. Genera of ticks represented by blue for Ixodes sp., orange for Dermacentor sp., violet for Haemaphysalis sp. and green for Amblyomma sp.
The study produced the first analysis of host identification for local ticks that identifies animals to the species level by detecting their Cyt b gene in engorged on-host ticks. The usefulness of blood meal analysis in determining the identity of the host species by PCR has been demonstrated by a recent study (
In this study, the Cyt b gene was successfully proven as a discriminatory molecular marker for the identification of host DNA in ticks. Cyt b was selected as the target gene because of its track record in blood meal identification assays and its utilization in developing mammalian phylogeny (
The most significant aspect of the method is its sensitivity to detect minuscule amounts of host DNA. In this study, host DNA could be detected in the 27 engorged ticks as determined by electrophoresis on agarose gel. The successful identification presumably because ticks sample was in the freshly engorged state, sufficient intact host DNA was present in the midgut. Adult stage of the ticks was used in this analysis may also eventually give positive identification of host.
This study shows that Ixodes granulatus may probably pose greater problems than most other ticks because of its capability to infest various hosts such as rodents and larger animals. This finding is in accordance with studies that reported I. granulatus as the most notable tick infesting rodents (
An interesting finding was observed in this study where Ixodes ticks (SBN 14) collected from R. tiomanicus gave a high similarity (99%) of blood meal to P. bengalensis. It was probably due to incomplete or an interruption of feeding that occurred before the tick attached itself to another host (
The finding of T. indicus as one of the hosts for Amblyomma ticks (K1a, K4b and K5b) was rare because the ticks were generally host specific on wild reptiles and amphibians (
Partial Cyt b mtDNA gene sequences used in this study seems to be effective in identifying the phylogenetic relationships between ticks and their hosts. This is because both NJ and MP tree topology showed very clear distinction between groups of small rodents, shrews and large mammals with a highly supported monophyletic clade. In small rodents, the node is solved in the NJ and MP tree as two monophyletic clades, representing the species of L. sabanus, R. tiomanicus and S. muelleri. The tree topologies also show that T. glis formed their own distinct monophyletic clade separated from other larger mammals which consists of P. bengalensis and T. indicus. The information obtained has further corroborated the morphological identification and classification of T. glis which comes under the group of shrews. Such knowledge gained through host preference studies is essential to understanding the relationship of host and vector and their roles in the enzootic transmission cycle (
Of particular interest was the high numbers of ticks infesting small rodents compared to larger mammals. This finding is in agreeable with a previous study which reported that the abundance of small rodents compensate even if the intensity of tick parasitism on them is smaller than the larger hosts (
For further studies, it is recommended that ticks are collected from sites frequently visited by animals including the wallows, wildlife main trails and river banks to include a more diverse group of animals in order to generate better results (
The PCR direct sequencing system using vertebrate Cyt b gene is a potential screening tool for the identification of ticks’ blood meal. The Cyt b gene was selected for this study based on their higher nucleotide variations for the effective identification of hosts. Blood meal identification of field collected ticks by molecular methods offer a direct and efficient approach for understanding the contributions of both competent and incompetent hosts for the transmission dynamics of tick-borne diseases. Furthermore, this valuable information can confirm a strong association between hard ticks and hosts (especially rodents) and this will assist public health officials with efforts to outline an effective tick-borne diseases control program.
The authors wish to thank the Director-General of Health, Malaysia, for permission to publish this paper. We wish to thank staff of the Acarology Unit, IMR for their assistance in the field. For the revision of English of the manuscript, we thank Serina Abdul Rahman. The study was supported by National Institute of Health grant (Code: JPP-IMR 11-010) from the Ministry of Health, Malaysia and ERGS/1/2011/STWN/UKM/03/9.