Nucleotide specific changes in the hypervariable region of 16S rDNA gene as possible marker to differentiate the tick genera.
Hyper variable segment of mitochondrial 16S rDNA from different stages of laboratory reared, disease free and acaricide susceptible Hyalomma anatolicum and Rhipicephalus (Boophilus) microplus were partially amplified, sequenced and analyzed with the aid of the GenBank database. Thirty conserved genus specific nucleotide change were observed in Hyalommid and Boophilid ticks. These conserved sequences were sufficient to identify embryonic stages of the ticks. These conserved sequences at the genus level could act as biomarker for identification of ticks during epidemiological studies of tick borne diseases, transmitted by Hyalommid and Rhipicephalid ticks.
Present address: 1,2Ph.D. student (rineshsinha @redifmail.com;
firstname.lastname@example.org; swetag009 @gmail.com), 8,9Principal
Scientist (dd_rayvetv @yahoo.co.in; email@example.com),
Entomology Laboratory, Parasitology Division, National
Agriculture Innovation Project; 7Senior Scientist (pallav.chaudhuri
@gmail.com), Division of Bacteriology and Mycology.
6Scientist and Head (firstname.lastname@example.org),
Pharmacognosy and Ethnopharmacology Division, National
Botanical Research Institute, Lucknow, Uttar Pradesh, India.
Indian Journal of Animal Sciences 81 (12): 1204–1207, December 2011
Detection of specific nucleotide changes in the hypervariable region of 16S rDNA
gene of Rhipicephalus (Boophilus) microplus and Hyalomma anatolicum anatolicum
Rinesh Kumar1, Souvik Paul2, Sachin Kumar3, Anil Kumar Sharma4, Sweta Gupta5, Ajay Kumar Singh Rawat6, Pallab
Chaudhuri7, D D Ray8 and Srikant Ghosh9
Indian Veterinary Research Institute, Izatnagar, Uttar Pradesh 243 122 India
Received: 9 December 2010; Accepted: 21 May 2011
Hyper variable segment of mitochondrial 16S rDNA from different stages of laboratory reared, disease free and
acaricide susceptible Hyalomma anatolicum anatolicum and Rhipicephalus (Boophilus) microplus were partially
amplified, sequenced and analyzed with the aid of the GenBank database. Thirty conserved genus specific nucleotide
change were observed in Hyalommid and Boophilid ticks. These conserved sequences were sufficient to identify
embryonic stages of the ticks. These conserved sequences at the genus level could act as biomarker for identification of
ticks during epidemiological studies of tick borne diseases, transmitted by Hyalommid and Rhipicephalid ticks.
Key words: Hyalomma anatolicum anatolicum, Rhipicephalus (Boophilus) microplus, 16S rDNA
Hyalomma anatolicum anatolicum and Rhipicephalus
(Boophilus) microplus are considered to be the most
damaging tick species in India, and the control program
against tick-borne diseases is specifically focused on these
tick species (Ghosh et al. 2007). These tick species are being
maintained in the entomology laboratory of the institute
(Azhahianambi and Ghosh 2007) and are under use for
various entomological, immunological, biochemical and
Molecular techniques were used for genus level
identification of several ticks (Black and Piesman 1994,
Crampton 1996, Black et al. 1997). The genes were preferred
in comparison to ITS1 ITS2 and 18s rDNA segments because
the sequences are shorter and more consistent in length, have
a higher percentage of alignable bases for sequence
comparisons and devoid of any complications caused by the
multiple alleles or introns (Mangold 1998). An important
aspect of rDNA of ticks is that they have secondary structures
that are moderately well conserved among distantly related
taxa (Caetano-Anollés 2002), while hypervariable regions
of 450bp is widely variable in different tick species. The
hypervariable region is targeted for identification of genus
specific conserved nucleotides by amplification and
sequencing of this region. Universal primers are usually
chosen as complementary to the conserved regions at the
beginning of the gene and at either the 540–bp region or at
the end of the whole sequence (about the 1,550 bp region),
and the sequence of the variable region in between is used
for the comparative study.
The aim of current study was to characterize hypervariable
segments of the 16S rDNA in hyalommid and rhipicephalid
ticks and to identify the specific regions of the hypervariable
segment that would be most characteristic for identifying
genera specific nucleotide changes amongst the entire tick
species study set. The possibility of using the genus specific
nucleotide changes as marker to differentiate the tick genera
MATERIALS AND METHODS
Tick material: Larvae, partially fed adults, and unfed
adults of 3 different generations of laboratory maintained
colony of H anatolicum anatolicum and adult of R (B)
microplus were used in the experiment. The different stages
of ticks were maintained by rearing on cattle and rabbit hosts
as per Ghosh and Azahianambi (2007). The animals were
maintained as per the approved guidelines of the Committee
December 2011] NUCLEOTIDE CHANGES IN RHIPICEPHALUS MICROPLUS AND HYALOMMA A. ANATOLICUM 1205
for Protection, Supervision and Control of Experiments on
Animals (CPCSEA), a statutory Indian body monitoring the
use of animals in experiments.
DNA extraction and PCR amplification: DNA was
extracted from pooled larvae, partially fed adults and unfed
adults of laboratory reared H. anatolicum anatolicum ticks
and adults of R.(B.) microplus for amplification and
sequencing. Universal primers were used for the
amplification of 450bp hypervariable region at 3′ end of 16S
rDNA gene. The forward primer and reverse primer used
were 5′- CCGGTCTGAACTCAGATCAAGT-3′ and 5′-
originally designed by Norris et al. (1996) The PCR
conditions was optimized using PTC100 model DNA engine
and all the reagents were procured commercially. For 25 µl
PCR master mix, the concentration of the reagents were
Truestart Hotstart buffer (10X) 2.5 µl, dNTPs (10 mM) 0.5
µl, MgCl2 (25 mM) 2 µl, forward primer (10 pM) 1 µl, reverse
primer (10 pM) 1 µl, Truestart Hotstart Taq polymerase
enzyme (5 U/µl) 0.3 µl, template DNA 5.0 µl, nuclease free
water 12.7 µl. The PCR amplifications were carried out as
follows: an initial denaturation step of 3 min at 94°C,
followed by 32 cycles, each of denaturation for 1 min at 94°C,
primer annealing for 1 min at 50.5°C and primer extension
for 1 min at 72°C. A final extension step was carried out for
15 min at 72°C. Negative controls (no template) were run
simultaneously, and reaction mixtures were discarded when
any DNA appeared in the control. A 10µl of the reaction
mixture was examined in 1.5% agarose gel electrophoresis
followed by staining with ethidium bromide, to check the
size of amplified fragments by comparison to a DNA
molecular weight marker.
DNA sequencing: PCR products were sent to ILS
Bioscience and Department of Biochemistry, New Delhi
India, for double strand sequencing and confirmation of the
sequence information. In direct sequencing of PCR products,
the entire gene was sequenced in both the directions.
Sequence analysis: Six Hyalomma and 4 Boophilus 16S
rDNA sequences were aligned with available data base of R
(B) microplus in the GenBank. using CLUSTAL V method
(Higgins et al. 1992). No sequence of H. anatolicum
anatolicum was available in the GenBank database.
RESULTS AND DISCUSSION
PCR amplification of mitochondrial 16S rDNA from
acaricide susceptible laboratory reared H. anatolicum
anatolicum and Rhipicephalus (B.) microplus species yielded
a fragment of 449 bp and of 454 bp, respectively. The
amplified product was directly used for sequencing after
purification. To avoid any reading error, both the strands were
sequenced. The GenBank accession numbers for these
sequences are as follows:Hm176656, Hm176658,
Hm176657, GU222462, GU323287, GU323288.
The nucleotide base composition of the sequenced rDNA
hypervariable region showed a high A+T content. It was
approximately 77% for all the sequences reported in this
study. The average base composition was 39.2% adenine,
9.6% cytosine, 13.8% guanine and 37.4% thymine.
A high sequence similarity of 16S rDNA (99.8% or more)
was observed between the different stages of the colony of
acaricide susceptible laboratory reared H. anatolicum
anatolicum tick species. Alignment of sequence data showed
that 30 nucleotides across this hypervariable region of
mitochondrial 16S rDNA were conserved in Hyalomma
species while the corresponding nucleotides in Rhipicephalus
species were different but conserved among Rhipicephalus
species (Table 1). The nucleotide substitution between
Hyalomma and Rhipicephalus species were found to be
transversion. The most frequent transversions were of A”!T
type (70% of the total substitution).
Ribosomal DNA (rDNA) is the most conserved region of
the genome in all the cells. In this study mitochondrial
ribosomal 16S DNA sequences were used as an essential
tool for identifying and classifying the tick genera. We
amplified and reported the first 3 sequences of ribosomal
16S rDNA of H. anatolicum anatolicum from different stages
of the laboratory reared, acaricide susceptible, homogenous
tick colony (Accession nos- Hm176656, Hm176657,
Hm176658). In Hyalomma, out of 30 genus specific
nucleotide, 43% T, 33% A, 13% C, and 10% G, while in
Rhipicephalus, the corresponding nucleotides were — 33%
T, 47% A, 7% C, and 13% G. The high percentage of
similarity (99.8% or more) as observed between the different
stages of the laboratory reared, H anatolicum anatolicum
and R. (B.) microplus could be considered as an indicator of
genetic homogeneity in the colony of laboratory reared tick
species. These tick species is adapted in the laboratory and
is continuously rearing for the last 10–12 years. This inbred
population maintained genus-specific nucleotide
conservation in the hypervariable region along with other
species of the genus.
Sequence analysis of conserved housekeeping genes such
as the 16S rRNA are increasingly being used for phylogenetic
studies (Black and Piesman 1994, Crampton 1996, Black et
al. 1997, Mangold 1998) but in the present study
mitochondrial ribosomal 16S DNA sequences are used as an
essential tool for identifying and classifying the tick genera
as reported by Labruna et al. (2009). The research
laboratories where number of tick species are maintained
for conducting different types of experiments involving
specific vectors and pathogens, a molecular tool for
identification of the tick genera at embryonic level will be
of great importance. In the epidemiological study, many times
embryonic stages of the ticks are collected and the same can
be processed and sequenced for comparison with the known
sequences before using the sample for isolation of pathogens.
Since the tick species cannot be differentiated morpho-
logically at embryonic level, the molecular tool can be a
1206 KUMAR ET AL. [Indian Journal of Animal Sciences 81 (12)
useful aid for differentiation of species and save the crucial
biological material. This method is highly sensitive and since
the method is based on mitochondrial DNA amplification,
degraded or small amounts of samples can also be analyzed.
The universal primer set enables PCR amplification of 16S
rDNA sequences in the samples from many tick species for
direct sequencing (Norris et al. 1996) and the sequences can
Table 1. Conservation of nucleotides in Hyalomma and Boophilus genera
1 90 92 96 99 113 125 141
TT A AATC H. a. anatolicum (LHa) Hm176656
TT A AATC H. a. anatolicum (UFH) Hm176658
TT A AATC H. a. anatolicum (PFH) Hm176657
TT A AATC H. dromedari L34306
TT A AATC H. lusinaticum Z97881
TT A AATC H. marginatum L34307
AC G TTAT R.(B.) microplus (34th gen) GU222462
AC G TTAT R.(B.) microplus (35th gen) GU323287
AC G TTAT R.(B.) microplus (37th gen) GU323288
AC G TTAT R.(B.) annulatus Z97877
186 188 190 191 224 227
AG TTCG H. a. anatolicum (LHa) Hm176656
AG T TCGH. a. anatolicum (UFH) Hm176658
AG. T T CGH. a. anatolicum (PFH) Hm176657
AG TTCG H. dromedari L34306
AG T TCGH. lusinaticum Z97881
AG T T CG H. marginatum L34307
TA A ATAR(B.) microplus (34th gen) GU222462
TA A ATAR.(B.) microplus (35th gen) GU323287
TA A ATAR.(B.) microplus (37th gen) GU323288
TA AATA R. (B.) annulatus Z97877
237 239 252 263 267 271 277 287
AC T ATTT A H. a. anatolicum (LHa) Hm176656
AC T ATTT A H. a. anatolicum (UFH) Hm176658
AC T ATTT A H. a. anatolicum (PFH) Hm176657
AC T ATTT A H. dromedari L34306
AC T ATTT A H. lusinaticum Z97881
AC T ATTT A H. marginatum L34307
G T A T G A C G R.(B.) microplus (34th gen) GU222462
G T A T G A C G R.(B.) microplus (35th gen) GU323287
G T A T G A C G R.(B.) microplus (37th gen) GU323288
GT A TGAC G R. (B.) annulatus Z97877
290 92 299 323 327 341 353 400
CTA T A A T T G H. a. anatolicum (LHa) Hm176656
CTA T A A T T G H. a. anatolicum (UFH) Hm176658
CTA T A A T T G H. a. anatolicum (PFH) Hm176657
CTA T A A T T G H. dromedari L34306
CTA T A A T T G H. lusinaticum Z97881
CTA T A A T T G H. marginatum L34307
AAT A T T A A A R.(B.) microplus (34th gen) GU222462
AAT A T T A A A R.(B.) microplus (35th gen) GU323287
AAT A T T A A A R.(B.) microplus (37th gen) GU323288
AAT A T T A A A R.(B.) annulatus Z97877
LHa- larvae, UFH- unfed adult, PFH- partially fed adult of Hyalomma.
be compared with a great stock of registered data in the
GenBank. The method can be effectively used in the
laboratories working on the epidemiological studies of fatal
tick borne human and animal diseases transmitted by
Hyalomma and Rhipicephalus tick species.
We are in the process of analyzing a large pool of sequence
data generated in our laboratory which will help in
December 2011] NUCLEOTIDE CHANGES IN RHIPICEPHALUS MICROPLUS AND HYALOMMA A. ANATOLICUM 1207
development of biomarker for identification of tick genera
at embryonic stage.
Authors are grateful to ICAR, New Delhi for providing
funding support through World Bank funded NAIP Project
no. NAIP/Comp-4/C2066/2008–09, C2066.
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