823-Article Text-3390-2-10-20190624

J Arthropod-Borne Dis, June 2019, 13(2): 153–164 EM Abouelhassan et al.
: Comparison of some …
Original Article
Comparison of some Molecular Markers for Tick Species Identification
*Eman M. Abouelhassan1; Hamdy M. ElGawady1; Ahmad Anwar AbdelAal1; Amal K. El-
Gayar1; Maria D Esteve-Gassent2
1
Department of Veterinary Parasitology, Suez Canal University, Ismailia, Egypt
2
Department of Veterinary Pathobiology, Texas A and M University, College Station, Texas, United
States of America
(Received 15 Mar 2018; accepted 22 Apr 2019)
Abstract
Background: Ticks are obligate blood-sucking ectoparasites of vertebrates. Since many tick identification studies
are based on the analysis of 16S rDNA, 12S rDNA and ITS-1, 2 rDNA genes, we aimed to compare the performance
of these molecular markers of common use for the identification of ticks, under a diagnostic laboratory environment.
Methods: Overall, 192 tick specimens were collected through the state of Texas from January 2014 to August 2015
and the species was determined by both morphology and molecular amplification using the 16S rDNA, 12S rDNA,
ITS1 and ITS2.
Results: The species collected were identified by molecular techniques as Dermacentor albipictus, D. variabilis, Am-
blyomma americanum, Ixodes scapularis, A. cajennense, Rhipicephalus sanguineus and Carios capensis. ITS1 and ITS2
were not able to prove consistent amplification and therefore have been considered as potential markers for tick iden-
tification.
Conclusion: The use of mitochondrial genes in tick identification showed to provide more consistent results in the
diagnostic environment.
Keywords: Ticks; Dermacentor; Amblyomma; Rhipicephalus; Ixodes
Introduction
Ticks are obligate blood-sucking ectopar- tious diseases (2) such as anaplasmosis, ehr-
asites of vertebrates, causing great economic lichiosis, and lyme borreliosis are transmitted
losses to livestock with its direct and indirect by ticks.
effects on hosts. Bloodsucking by large num- Table 1 shows the pathogens transmitted
bers of ticks causes a reduction in live weight by different species of ticks as Ixodes species
and anemia among domestic animals, while are the vectors of lyme borreliosis, Amblyomma
their bites also reduce the quality of hides (1). americanum is the vector of Ehrlichia chaffeen-
In addition, certain ticks will cause tick paral- si, tick identification helping in the diagnosis
ysis, which is an acute ascending flaccid mo- of disease transmitted with, and misidentifi-
tor paralysis caused by the injection of a tox- cation of the may lead to difficult and even
in by the tick while feeding. However, the wrong disease diagnosis.
major effects caused by ticks are due to their Even though there is a lack of whole tick
ability to transmit protozoan, bacterial and viral genome annotation, molecular techniques in
diseases to livestock, companion animals and acarology have been made available in the
humans (16, 17). Ticks are currently consid- past few years (9), traditionally tick identifi-
ered to be second only to mosquitoes as vec- cation has always been based on morpholog-
tors of human infectious diseases in the world ical characteristics. Moreover, to identify the
(9, 23). A number of bacterial zoonotic infec- immature tick stages (larvae, nymph and adults)
153
*Corresponding author: Dr Eman Mohammed http://jad.tums.ac.ir
Abouelhassan, E-mail: [email protected] Published Online: June 24, 2019
J Arthropod-Borne Dis, June 2019, 13(2): 153–164 EM Abouelhassan et al.: Comparison of some …
separate keys are normally used (2, 3). In cer- arthropods. One of the limitations is the large
tain situation, damage to tick body parts essen- size of tick genomes (15). For instance, the
tial for their identification (such as capitulum average haploid genome of the tick I. scapu-
and adjacent structures) may occur during re- laris has been calculated at 2262Mbp in length,
moval of attached ticks to their hosts. In ad- while the A. americanum is around 3108Mbp.
dition, bad preservation of tick samples often If we compare this to the human genome, tick
occurs, leading to incorrect identifications (4). genomes tend to be twice as bigger as the hu-
Identification of some tick species like Rhip- man genome. Part of the difference in size is
icephalus sanguineus and Amblyomma cajen- due to the presence of non-coding regions with
nense is difficult due to the fact that they have extended tandem repeats, that difficulty signif-
been classified as a species complex (11, 12). icantly the sequencing and annotation of those
For instance, A. cajennense is a complex of 6 genomes (15). Consequently, different molec-
species, while R. sanguineus group compris- ular markers have been traditionally used for
es a total of 17 different species. In these com- the phylogeny of ticks (8). Those include the
plexes, the different species are geographically nuclear ribosomal genes 18S rDNA, 28S rDNA
separated, due to the large geographical range and ITS-1, 2 rDNA as well as mitochondrial
of distribution, and the expected adaptation genes such as 16S, 12S, COI, COIII) rDNA
of tick populations to different environmen- (8, 9).
tal conditions (12). Therefore, morphological El-Fiky and El Kammah (3) and Chitimia
identification of these species is not sufficient (4) successfully used the internal transcribed
and the further molecular information is need- spacer (ITS) for the identification of Derma-
ed for a correct species determination (11, 12). centor marginatus, Ixodes ricinus, Haema-
These difficulties may be reduced when physalis, Boophilus, and Rhipicephalus san-
using molecular techniques for tick identifi- guineus tick species. On the other hand, the
cation (3). Another benefit from molecular tech- 16S rDNA were able to construct the phy-
niques is that with those samples that tick DNA logeny of both hard and soft ticks (6, 8, 9).
integrity has not been compromised, from the 16S rDNA were used in molecular classification
total DNA extraction, a collection of different of metastriate ticks (Dermacentor, Amblyom-
tick-borne pathogens can be detected by molec- ma and Rhipicephalus respectively (10, 13).
ular methodologies such as conventional PCR, Since many tick identification studies are
and real-time PCR (4). Therefore, with one sin- based on the analysis of 16S rDNA, 12S rDNA
gle extraction, both the agent and the vector and ITS-1, 2 rDNA genes, the objective of the
species can be determined (4). Moreover, the present study was to compare the performance
availability of genetic sequence will provide of these molecular markers of common use
the opportunity to study both the vector pop- for the identification of ticks, under a diag-
ulation diversity, as well as the pathogen they nostic laboratory environment (13).
carry, and potentially even their relationships
(13). Currently, there is a lack of whole tick
genomes readily annotated Materials and Methods
(https://www.vectorbase.org/), with Ixodes
scapularis being the only one currently avail- Tick sample collection
able Overall, 192 specimens were utilized in
(https://www.vectorbase.org/organisms/ixod this study. This collection was divided into two
es-scapularis) (13). This lack of information groups, group A comprises 59 ticks (larvae,
limits the advancement of the development of nymph, males and engorged females) obtained
new molecular methods for the study of these from Texan citizens through the tick testing
154
http://jad.tums.ac.ir
Published Online: June 24, 2019
service provided by the Lyme Laboratory at the mixture. The mixture was run through fil-
Texas A and M University, from January 2014 ter columns at 13,000×g for 3min. DNA bound
to August 2015. On the other hand Group, B to filter was washed and eluted following man-
contains 133 tick pools collected from wild- ufacturer recommendations. To extract DNA
life through an ecology project conducted in from the tick immature stages (nymphs and
collaboration with Dr. Castro-Arellano at Tex- larvae) pools of a maximum of 15 nymphs or
as State University (Table 2). For better DNA 50 larvae were made. Specimens received a
extractions, larvae and nymphs were analyzed code indicating the type of pool generated.
in pools, sometimes the pools contain one lar- All immature specimens were stored at -80
vae and other up to 50 larvae of the same tick ºC with 100µl TE buffer for at least one
species collected from the same location in the hour. Specimens were homogenized utilizing
same sampling effort. Nymphs, on the other pestles while the samples were frozen, fol-
hand, were pooled in groups of from one till 15 lowed by DNA extraction procedures using
specimens, following the same strategy de- the prepGEM™ (ZyGem Ltd., New Zeland)
scribed for larvae for optimal DNA extraction. Insect DNA Extraction kit following manu-
For the majority of these ticks, the morpho- facturer’s recommendations. Briefly, tick sam-
logical identification was not enough to de- ples were mixed with ultra-pure water, 10x
termine the species, mostly due to either bad buffer provided in the kit, and 1µl of the
storage of samples or loss of mouth-parts while prepGEM™ enzyme (ZyGem Ltd., New
removal the tick from the host. These ticks were Zeland). The mixture was incubated at 75 °C
immersed in 70% ethanol solution and then for 15min followed by incubation at 95 °C
processed for DNA extraction and molecular for 15min. The extracted DNA concentration
identification using 16S rDNA PCR specific and purity were measured using a NanoDrop,
primers and sequencing the PCR product. and stored at -20 until use.
DNA Extraction Molecular identification of ticks based on

DNA was extracted from the tick samples 16S rDNA Gene, 12S rDNA and ITS-1 and
using Wizard® SV Genomic DNA Purifica- 2 rDNA Genes
tion kit (Promega Corporation, Madison, WI) The tick 16SrDNA was amplified from
following manufacturer’s recommendations each specimen studied using conventional PCR
with modifications. Briefly, ticks were incu- methodologies and utilizing primers (6) (Ta-
bated for 10min at 70 ºC in 200µl of Nuclei ble 3) and AccuPrime™ SuperMix (Quanta Bi-
Lysis Solution, plus 50µl of 0.5M EDTA, 40 oscienceInc., Gaithersburg, Maryland). The
µl of a 20mg/ml Proteinase K solution, and PCR was run following the cycling condi-
5µl RNase A Solution. After the initial diges- tion: initial denaturation at 95 ºC for 5min
tion and for the optimal extraction of DNA followed by 10 cycles of 92 ºC for 1min, 48 ºC
from the arthropods, adult individual ticks were for 1min and 72 ºC for 90sec, this step was
homogenized utilizing the bead mill Bead Rup- followed by additional 32 cycles of 92 ºC for
tor 24 (Omni International, Inc., Kennesaw, 1min, 54 ºC for 35sec and 72 ºC for 90sec,
GA), un-engorged ticks were homogenized this was followed by a final extension at 72
with 1.4mm ceramic beads while 2.8mm ce- ºC for 7min (5). The amplification products
ramic beads were used with engorged ticks. Af- from 16S rDNA were separated on 1.6% aga-
ter homogenization, tubes were centrifuged rose gel containing 0.4µg/ml of ethidium bro-
at 10,000×g to eliminate tick debris. Superna- mide (Bio-Rad Laboratoies Inc., Hercules, CA)
tants were collected and 250µl of Wizard® at 90 volts for 40–60min, and imaged using
SV Lysis Buffer was added to each sample and ChemiDoc touch imaging system (Bio-Rad La-
155
boratoies Inc., Hercules, CA). Positive bands sent for sequencing (Eton Biosciences, San
were excised from the gel and purified using Diego, CA). Sequences were analyzed through
the Wizard® SV Gel and PCR clean-up sys- BLAST® using MacVector 14.0 software
tem (Promega Corporation, Madison WI) fol- (MacVector Inc., Cary, NC).
lowing manufacturer’s recommendations. The
purified products were sent for sequencing
Results
(Eton Biosciences, San Diego, CA). Sequences
were analyzed through BLAST® in MacVec-
Overall, 192 ticks were analyzed from dif-
tor 14.0.0 software (MacVector Inc., Cary, NC).
ferent developmental stages (larvae, nymph,
PCR was performed first using16S rDNA
males and engorged females) collected through
Gene primers. The same samples were also
the state of Texas. In this collection, the tick
tested using specific primers for first and sec-
specimens were identified based on the16S
ond internal transcribed spacers (ITS-1 and
rDNA PCR products as Dermacentor albipic-
ITS-2 rDNA) (Table 3) following methodol-
tus, D. variabilis, Amblyomma americanum,
ogies (4). The PCR reaction was done using
Ixodes scapularis, A. cajennense Rhipiceph-
the following cycling condition: initial dena-
alus sanguineus and Carios capensis (Table
turation at 95 ºC for 5min followed by forty
4), the GenBank accession numbers from
cycles of 95 ºC for 45sec, 55 ºC for 1min and
KX673167 to KX673180.
72 ºC for 90sec with a final extraction at 72 ºC
for 1min. The amplification products were sep-
Comparison between 16S rDNA Gene, 12S
arated on 1.6% agarose gel containing 0.4µg/
rDNA Gene and (ITS-1, 2) rDNA Genes
ml of ethidium bromide (Bio-Rad Laboratoies
In order to evaluate which genetic mark-
Inc., Hercules, CA) and the gel was run at 90
er perform best under diagnostic conditions,
volts for 40–60min. Gels were visualized us-
comparison between the amplification of four
ing the ChemiDoc touch imaging system (Bio-
genes 16S rDNA, 12S rDNA, ITS1 and ITS2
Rad Laboratoies Inc., Hercules, CA).
rDNA genes was done. Positive bands from
In 12S rDNA, PCR was done following
16S rDNA, 12S rDNA PCR were excised from
the cycling condition: initial denaturation at
the gel and cleaned and submitted for se-
95 ºC for 5min followed by forty cycles of 95
quencing. Sequences were analyzed through
ºC for 30sec, 40 ºC for 30sec and 72 ºC for
BLAST® in MacVector 14.0.0 software
30sec, with a final extraction at 72 ºC for 5
(MacVector Inc., Cary, NC).
min. The amplification products were sepa-
in spite of the samples number are not rep-
rated and visualized as mentioned before on
resentative, but changing in the tick PCR am-
1.6% agarose gel containing 0.4µg/ml of eth-
plification of the four genes was observed as
idium bromide (Bio-Rad Laboratoies Inc., Her-
good amplification for the samples were ob-
cules, CA) and the gel was run at 90 volts for
served as both 16S rDNA, 12S rDNA Gene
40–60min. Gels were visualized using the
showing good PCR amplification in all the
ChemiDoc touch imaging system (Bio-Rad
sample but (ITS-1, 2) rDNA Genes failed to
Laboratoies Inc., Hercules, CA).
amplify some samples species, (Fig. 1).
The samples utilized were Dermacentor
Sequence analysis
albipictus (CVM11, CETX-2), Dermacentor
Positive bands were excised from the gel
variabilis (ARCO1), Amblyomma american-
and purified using the Wizard® SV Gel and
um (MMSL5, MMSL9), Ixodes scapularis
PCR clean-up system (Promega Corporation,
(CSTX-2, CSTX-3), Amblyomma cajennen-
Madison WI) following manufacturer’s rec-
se (BAS 115) and Rhipicephalus sanguineus
ommendations. The purified products were
156
(SAT 88, SAT 97, and SAT 101).

In ITS-1PCR, one of the three samples of
Rhipicephalus sanguineus (SAT 101), two sam-
ples of Amblyomma americanum (MMSL5,
MMSL9), Ixodes scapularis samples CSTX-
2, CSTX-3) and Dermacentor albipictus sam-
ples (CVM11, CETX-2) were amplified, but
there were no amplification in Dermacentor
variablis (ARCO1), Amblyomma cajennense
(BAS 115) and the other two samples of R.
sanguineus (SAT 88, SAT 97).
ITS-2 PCR, one of the three samples of
Rhipicephalus sanguineus (SAT 101), two sam-
ples of Amblyomma americanum (MMSL5,
MMSL9), Dermacentor variablis (ARCO1),
Amblyomma cajennense (BAS 115), Ixodes
scapularis samples CSTX-2, CSTX-3) and
Dermacentor albipictus samples (CVM11,
CETX-2) were amplified, but the two samples
of R. sanguineus (SAT 88, SAT 97) failed to
amplify. Fig. 1. PCR amplification utilizing Tick samples of
different species using: (A) the ITS-2PCR reaction
Phylogenetic analysis (B): ITS-1 PCR reaction and (C): 16S r DNA Gene,
The phylogenetic analysis was performed (D)12SrDNA DNA ladder is located on the left and
using MacVector 14.0 software (MacVector right sides of the gel, fragment sizes are represented
in base pairs (bp), 1: Rhipicephalus sanguineus sam-
Inc., Cary, NC) and the tree was constructed ple (SAT 97), 2: Rhipicephalus sanguineus sample
using neighbor-joining (NJ) methods (Figs. (SAT88), 3: Rhipicephalus sanguineus sample (SAT
2-4). The low degree of sequences variation 101), 4: Amblyomma americanum sample (MMSL5),
observed within most of the species of the soft 5: Amblyomma americanum sample (MMSL9), 6
and hard tick trees based on the 16S rDNA Amblyomma cajennense (BAS 115 TICK): 7: Ixodes
scapularis sample (CSTX-2), 8: Ixodes scapularis
since they all share the same ancestor. Nev- sample (CSTX-3), 9: Dermacentor albipictus sam-
ertheless, there is one sequence in the hard tick ple(CVM11), 10: Dermacentor albipictus sam-
population studied Rhipicephalus sanguineus ple(CETX-2), 11: Dermacentor variabilis sam-
that show higher variation. ple(ARCO1) and 12: negative control.
Table 1. Important tick-borne diseases of humans

Pathogens Disease Vectors Distribution Reference
Borrelia burgdorferi Lyme borreliosis Ixodes ricinus, I. Asia, Europe, 17,18,19
senso lato pacificus, I. scapu- North America
laris, I. hexagonus
Ehrlichia canis Human Ehrlichiosis Rhipicephalus South America, 19
sanguineus Asia, Africa
Ehrlichia ewingii Human ewinigii Amblyomma amer- USA, Africa, 20
ehrlichiosis icanum Asia
Ehrlichia muris Murine splenomeg- Haemaphysalis Eurasia 20
aly spp, Ixodes spp
157
Table 1. Continued …
Ehrlichia chaffeensis Human monocytic Amblyomma amer- North America 17,18,19

ehrlichiosis icanum
Ehrlichia ruminantium Heartwater in rumi- Amblyomma spp Africa, Carib- 20
nant bean
Rickettsia conorii Mediterranean spot- Rhipicephalus Africa, Asia, 17,18,19
ted fever sanguineus, R. Europe
turanicus
Coxiella burnetii Q fever Many species Africa, Asia, 17,18,19
Europe, North
America, Aus-
tralia
Rickettsai rickettsii Rocky Mountain Amblyomma amer- North, South 17,18,19
spotted fever icanum, and Central
Rhipicephalus America
sanguineus, Der-
macentor variablis
Anaplasma phago- Human granulocytic Haemaphysalis North America, 17,18,19
cytophilum anaplasmosis concinna, H. punc- Europe
tate, Ixodes rici-
nus, I. pacificus, I.
scapularis
Rhipicephalus
bursa
Flavivirus Tick borne encepha- Ixodes ricinus, Asia, Europe 18,19
litis Haemaphysalis
concinna, H. punc-
tate
Babesia divergen, B. Babesiosis Ixodes ricinus, I. North America, 17,18,19
microti scapularis Europe
Fig. 2. The phylogenetic analysis was constructed using neighbor joining method, to construct the tick phylogenetic
tree of some of soft tick species sequences from the Genbank and our sequences samples are included based on 16S r
DNA sequences
158
Fig. 3. The phylogenetic analysis was constructed using neighbor-joining method, to construct the tick phylogenetic
tree of some of hard tick species sequences from the Genbank and our sequences samples are included based on 16S
r DNA sequences
159
Fig. 4. The phylogenetic analysis was constructed using neighbor-joining method, to construct the tick phylogenetic
tree of some of hard tick species sequences from the Genbank and our sequences samples are included based on 12S
r DNA sequences
Table 2. Ticks samples utilized in this study according to their distribution and stages
Distribution Adult female Adult male Nymph Larvae
Arroyo, Colorado 1
Mason Mountain 2
Brazos County, Texas 24 1 1
Jefferson County 2 2 1
Texas 2
San Antonio, TX 3
Gus Engeling WMA 2 5
Tejas Ranch 6
Chaparral WMA 4 10
Las Palomas WMA-Arroyo Colorado Unit 86 52
Total 28 3 99 74
Table 3. Primers using in PCR

Gene F-Primers R-Primers Ref
Tick 16S rDNA 5´-TTGGGCAAGAAGACCCTATGAA -3´ 5´- CCGGTCTGAACTCAGATCAAGT-3´ (5)
Tick 12S rDNA 5´-GAGGAATTTGCTCTGTAATGG -3´ 5´-AAGAGTGACGGGCGATATGT-3´ (21)
ITS-1rDNA 5´-TCATAAGCTCGCGTTGATT-3' 5´-AGCTGGCTGCGTTCTTCAT - 3' (3)
ITS-2rDNA 5´-CGAGCTTGGTGTGAATTGCA-3´ 5´-TCCCATACACCACATTTCCCG-3' (3)
160
Table 4. Ticks samples utilized in this study according to their distribution and species
No. Samples name Tick species Location Developmental stage

1 ARCO1 Dermacentor variabilis Arroyo, Colorado Larvae
2 MMSL7 Amblyomma americanum Mason Mountain Nymph
3 MMSL8 Amblyomma americanum Mason Mountain Nymph
4 CETX-2 Ixodes scapularis Texas adult female
5 LPTX-1 Rhipicephalus sanguineus Brazos County, Texas adult female
6 BAS115 Amblyomma cajennense Brazos County, Texas adult female
7 BBLC1 Amblyomma americanum Brazos County, Texas adult female
8 RITX-1 Ixodes scapularis Jefferson County adult female
9 WOTX-1 Ixodes scapularis Brazos County, Texas adult female
10 SG-1 Ixodes scapularis Brazos County, Texas adult female
11 NSTX-1 Ixodes scapularis Texas adult female
12 BETX-20 Ixodes scapularis Jefferson County adult female
13 SAT-88 Rhipicephalus sanguineus San Antonio, TX Nymph
14 KTTX-5 Dermacentor variabilis Brazos County, Texas adult male
15 KTTX-6 Dermacentor variabilis Brazos County, Texas adult female
16 BAS-183 Amblyomma maculatum Brazos County, Texas adult female
17 BAS-125 Dermacentor variabilis Brazos County, Texas adult female
23 BAS-124 Dermacentor andersoni Brazos County, Texas adult female
24 SAT-97 Rhipicephalus sanguineus San Antonio, Texas Nymph
25 SAT-101 Rhipicephalus sanguineus San Antonio, Texas Nymph
26 MTX1 Ixodes scapularis Brazos County, Texas adult female
29 BETX-16 Ixodes scapularis Brazos County, Texas adult female
31 BETX-18 Ixodes scapularis Brazos County, Texas Nymph
33 THREAD1 Ixodes scapularis Brazos County, Texas adult female
34 CSTX1 Ixodes scapularis Brazos County, Texas adult female
35 CVM11 Dermacentor albipictus Brazos County, Texas adult female
36 CETX-2 Dermacentor albipictus Brazos County, Texas adult female
37 TJM 305-5 Carios capensis Chaparral WMA Larvae
38 TJM 448-514 Carios capensis Las Palomas WMA - Arroyo Colorado Unit 50 Larvae
39 TJM 182-1 Carios capensis Chaparral WMA Nymph
40 TJM 448 Carios capensis Las Palomas WMA - Arroyo Colorado Unit 48 Nymphs
41 TJM 216.1 Carios capensis Chaparral WMA Nymph
42 TJM 308-12 Carios capensis Chaparral WMA 3 Larvae
43 TJM 596 Dermacentor variabilis Las Palomas WMA - Arroyo Colorado Unit 38 Nymphs
45 TJM112 Dermacentor variabilis Gus Engeling WMA 2 Larvae
46 TJM 355 Dermacentor variabilis Chaparral WMA Nymph
47 TJM 440 Dermacentor variabilis Las Palomas WMA - Arroyo Colorado Unit 1 Larvae
48 TJM 308-18 Dermacentor variabilis Chaparral WMA 3 Larvae
49 TJM 140-3 Amblyomma inornatum Gus Engeling WMA 3 Larvae
50 TJM 139 Amblyomma inornatum Gus Engeling WMA 2 Larvae
51 TJM 529 Dermacentor variabilis Las Palomas WMA - Arroyo Colorado Unit 1 Larvae
52 TJM 216 Amblyomma maculatum Chaparral WMA Nymph
Discussion
The present studies aimed to present good molecular marker for tick identification based
161
on DNA sequences to solve the problems with scapularis, D. variabilis, A. cajennense, Car-
morphological tick identification, and some- ios capensis and Rhipicephalus sanguineus
times the morphological identification is not (Table 3). Therefore, using 16S rDNA and
enough for detect the species so amplification 12S rDNA are good in molecular tick identi-
of the16S rDNA using it as a methods for fication of all these species utilized in our study.
tick genetic identification, and comparison be- The first and the second internal transcribed
tween the amplification of the the16S rDNA, spacers region of the nuclear ribosomal gene
12S rDNA, ITS-1 rDNA and ITS-2 rDNA for cluster (ITS-1, ITS-2), consist of three genes
the same samples species. 18SrDNA, 5.8SrDNA and 28SrDNA. These
16S rDNA and 12S rDNA Genes are a mi- three rDNA genes are transcribed making a
tochondrial ribosomal DNA gene, mtDNA con- single transcript of RNA separated by the ITS-
sidered one of the most commonly used genes 1 and ITS-2 regions (7). (ITS-1, ITS-2) con-
for molecular identification of ticks due to the sidered the fastest evolving DNA genes (9).
fact that it is relatively easy to work with them Because of these facts, the ITS-1, 2 rDNA
due to their higher copy number (8). In addi- are not good in amplification of some of the
tion, mtDNA sequences are a good phyloge- tick species in our study, they failed to am-
netic marker mostly for groups of organisms, plify some tick species as mentioned before.
diverged relatively, since mtDNA has a higher Internal transcribed spacer was used success-
rate of base substitution than most nuclear fully for the identification of Dermacentor mar-
markers (9). The problem for the mitochon- ginatus, Ixodes ricinus, Haemaphysalis, Booph-
drial gene is it can transfer to the nucleus lead- ilus, and Rhipicephalus sanguineus tick spe-
ing to error in the phylogeny after the ampli- cies (3, 4).
fication and sequencing (8). The difference Nevertheless, the problem with internal
between the 16S rDNA and 12S rDNA Genes transcribed spacer is that genes are evolving
that the evolution is faster in 12S rDNA Genes rapidly so in some species they failed to am-
(8). plify as reported before in ticks (8). ITS-2 was
Regarding tick species, 16S rDNA was utilized for identification Iranian hard tick
used and succeeded to construct phylogeny and ITS-2 failed to amplify some of his sam-
of both hard and soft ticks (6, 7, 8) and 16S ples. Therefore, these markers were mostly
rDNA is useful in constructing their tick phy- useful to study close related species (10). Even
logenetic tree, but there is a problem asso- some of our samples are closely related to each
ciated with 16S rDNA is that using this gene other as Rhipicephalus species and Derma-
alone is not sufficient for getting full resolu- centor and it failed to amplify some of them,
tion for the tree so the best way to solve it therefore, it is better to clone the PCR products
accompanied it with another gene like 12S and work with it as haplotypes, not individu-
rDNA (6, 8). We utilized these genes for di- als make the studies more expensive (8).
agnostic purpose only not for phylogeny, and
the problems are usually associated with the
phylogeny. Conclusion
Overall, 192 tick samples (larvae, nymph,
males and engorged females) were evaluated Molecular tick identification will help and
using16S rDNA PCR and 12S rDNA the PCR improve the disease diagnosis and choosing
positive bands have to be sequencing. The se- good genetic marker for diagnosis purpose as
quencing analysis determined that the tick spe- 16S rDNA and 12S rDNA markers is good
cies collected in the study were: Amblyomma as they give good amplification for our sam-
americanum, Dermacentor albipictus, Ixodes ple species, in spite of using (ITS-1, ITS-2)
162
are good for tick molecular identification for Vet Parasitol. 129: 139–147.
very closely related species but with our few 6. Black WC, Piesman J (1994) Phylogeny of
samples even they are not representative sam- hard- and soft-tick taxa (Acari: Ixodida)
ples it did not work with the closely related based on mitochondrial 16S rDNA se-
tick species. quences. Proc Natl Acad Sci USA. 91
(21): 10034–10038.
7. Difference Between Hard Ticks and Soft
Acknowledgements
Ticks:
Thanks to the Lyme Laboratory at Texas http://www.orkin.com/other/ticks/hard-
A and M University and Dr Maria D Esteve- soft-ticks-difference
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J Arthropod-Borne Dis, June 2019, 13(2): 153–164 EM Abouelhassan et al.

(Received 15 Mar 2018; accepted 22 Apr 2019)

Keywords: Ticks; Dermacentor; Amblyomma; Rhipicephalus; Ixodes

DNA Extraction Molecular identification of ticks based on

(SAT 88, SAT 97, and SAT 101).

Table 1. Important tick-borne diseases of humans

Ehrlichia chaffeensis Human monocytic Amblyomma amer- North America 17,18,19

Table 3. Primers using in PCR

No. Samples name Tick species Location Developmental stage

/Genome-Analysis-of-Major-Tick-and- 23. Philippe Parola, Didier Raoult (2001) Ticks

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