Deciphering the molecular phylogenetics of the Asian honey bee, Apis cerana and inferring the phylogeographical relationships using DNA barcoding

Abstract

The Asian honey bee, Apis cerana are honey producers and pollinators of cultivated crops and wild plants. They occur in Asia, from Afghanistan to China and from Japan to southern Indonesia. A. cerana have yellow stripes on their abdomen and are habituated to Indian plains. These are less aggressive and also display less swarming behavior. Here we report the partial sequence of cytochrome oxidase sub unit I gene (COI) of A. cerana (GenBank Accession No. KM230116) and its phylogenetic relationship. The COI gene sequence of A. cerana showed considerable variation with other Apis species. The COI DNA barcode developed in this study can be used for accurate species identification. The COI partial coding sequence of A. cerana showed 2.72% difference over 513 bp nucleotides to A. cerana isolated from Indonesia (GU191875) and 6.04% to A. cerana isolated from Japan (AF153105). A. cerana demonstrates the efficiency of the barcoding gene in discriminating global phylogeographical variants among the Apis species complex.

Full text

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Journal of Entomology and Zoology Studies 2014; 2 (4): 218-220
ISSN 2320-7078
JEZS 2014; 2 (4): 218-220
© 2014 JEZS
Received: 28-07-2014
Accepted: 14-08-2014
Rukhsana, K.
Research Scholar, Molecular Biology
Laboratory, Department of Zoology,
University of Calicut, Kerala, India
Akhilesh, V. P.
Research Scholar, Molecular Biology
Laboratory, Department of Zoology,
University of Calicut, Kerala, India
Sebastian, C. D.
Assistant Professor, Molecular
Biology Laboratory, Department of
Zoology, University of Calicut,
Kerala, India
Correspondence:
Sebastian, C. D
Assistant Professor, Molecular
Biology Laboratory, Department of
Zoology, University of Calicut,
Kerala, India
Deciphering the molecular phylogenetics of the
Asian honey bee, Apis cerana and inferring the
phylogeographical relationships using DNA
barcoding
Rukhsana, K., Akhilesh, V. P. and Sebastian, C. D.
Abstract
The Asian honey bee, Apis cerana are honey producers and pollinators of cultivated crops and wild
plants. They occur in Asia, from Afghanistan to China and from Japan to southern Indonesia. A. cerana
have yellow stripes on their abdomen and are habituated to Indian plains. These are less aggressive and
also display less swarming behavior. Here we report the partial sequence of cytochrome oxidase sub unit
I gene (COI) of A. cerana (GenBank Accession No. KM230116) and its phylogenetic relationship. The
COI gene sequence of A. cerana showed considerable variation with other Apis species. The COI DNA
barcode developed in this study can be used for accurate species identification. The COI partial coding
sequence of A. cerana showed 2.72% difference over 513 bp nucleotides to A. cerana isolated from
Indonesia (GU191875) and 6.04% to A. cerana isolated from Japan (AF153105). A. cerana demonstrates
the efficiency of the barcoding gene in discriminating global phylogeographical variants among the Apis
species complex.
Keywords: A. cerana, DNA barcoding, phylogeny, cytochrome oxidase.
1. Introduction
The Asian bee, Apis cerana (Hymenoptera: Apidae) is found throughout Asia and across a
diverse range of climatic zones
[1]
. The life cycle of Asian bees is very similar to that of Apis
mellifera and its life cycle completed within 21 days. The colony is structured with a single
fertile female (the queen) several thousand worker bees and seasonally, male bees.
Mitochondrial genomes are renowned for mutation hot spots or adaptive substitutions which
makes the genome more noteworthy, and results in the heterogeneous evolutionary rates across
genes
[2]
. The average rate of evolution of the mitochondrial genome is known to be 5-10 times
higher than that of nuclear genome, in case of mammals
[3]
. The focus of the current study is to
decipher the systematic position of A. cerana using mitochondrial and nuclear genes in the
order Hymenoptera
[4]
.
Studies on the biology and distribution of races of A. cerana in China were done
[5]
. A
morphological analysis of A. cerana and populations from Southeast Asia has also been taken
[6]
. Data on comparative morphology of the dwarf honey bees in Southeast Thailand and
Palawan, Philippines has been published
[3]
. Wongsiri et al. (1993) carried out a comparative
investigation of some biological characteristics of A. cerana bees in China, Thailand and their
hybrids for the purpose of using biological measures to control Varroa parasitic mites
[7]
.
There is a big gap in information on the genetic diversity of Asian native honey bees. The
purpose of this paper is to present some preliminary results on comparative genetic
composition of A. cerana with some honey bee species, to contribute basic information on
certain genetic parameters.
2. Materials and Methods
Asian honey bee, A. cerana used in the present study was collected from Kottakkal, Kerala on
21
st
October 2013. Mitochondrial genomic DNA was extracted from one of the thoracic legs of
the experimental insect, A. cerana. The tissue was homogenized using a glass pestle and
mortar. The genomic DNA in the homogenate was extracted using GeNei Ultrapure
Mammalian Genomic DNA Prep Kit (Banglore GeNei, Banglore). About 2 ng of genomic
DNA was amplified for mitochondrial cytochrome oxidase subunit (COI) gene using the

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Journal of Entomology and Zoology Studies
forward primer 5'-GGTCAACAAATCATAAAGATATTGG
-3' and the reverse primer 5'-
TAAACTTCAGGGTGACCAAAAAATCA -3'. The PCR
reaction mixture consisted of 2 ng of genomic DNA, 1 µl each
forward and reverse primers at a concentration of 2.5 µM, 2.5
µl of dNTPs (2 mM), 2.5 µl of 10X reaction buffer, 0.20 µl of
Taq polymerase (3 U/µl) and 11.8 µl H
2
O. The PCR profile
consisted of an initial denaturation step of 2 minutes at 95
°
C,
followed by 30 cycles of 5s at 95
°
C, 45s at 50
°
C and 45s at 72
°
C and ending with a final phase of 72
°
C for 3 minutes. The
PCR products were resolved on 1% TAE-agarose gel, stained
with Ethidium bromide and photographed using a gel
documentation system. After ascertaining the PCR
amplification of the corresponding COI fragment, the
remaining portion of the PCR product was column purified
using Mo Bio Ultraclean PCR Clean-up Kit (Mo Bio
Laboratories, Inc. California). The purified PCR product was
sequenced from both ends using the forward and reverse
primers used for the PCR using Sanger’s sequencing method
at SciGenom Labs Pvt. Ltd, Cochin. The forward and reverse
sequences obtained were trimmed for the primer sequences,
assembled by using ClustalW and the consensus was taken for
the analysis. The nucleotide sequence and peptide sequence
were searched for its similarity using BLAST programme of
NCBI (www.ncbi.nlm.nih.gov/) and Inter and intra specific
genetic diversity were calculated using Kimura 2-parameter
model with the pair wise deletion option and the difference in
the nucleotide in codon usage partial COI sequence of A.
cerana was analyzed using MEGA6 software.
3. Results and Discussion
DNA sequence based identification technique has revealed the
morphological and ecological traits of many species during
larval stages
[8, 9, 10]
. Gurney et al. (2000) reported that closely
related species have 90% similarity in the standardized DNA
sequence and distantly related species have less than 90%
similarity in the same genes sequence
[11]
. Intraspecific
divergence of partial coding fragment of COI gene is very
efficient for species identification
[11]
. The COI sequence of A.
cerana showed close similarity within the species and
considerable variation between the species. Therefore the COI
sequence of A. cerana can be used for the molecular
identification in any stage of life cycle. The PCR of the COI
gene fragment of A. cerana yielded a single product of 513 bp.
The BLAST search using the sequence revealed that the
sequence obtained in this study is novel. The partial COI DNA
sequence of A. cerana (GenBank Accession No. KM230116)
showed 2.72% difference with that of A. cerana (GenBank
Accession No. GU191875) isolated from Indonesia and
6.04% difference with A. cerana isolated from Japan
(GenBank Accession No. AF153105). The partial COI coding
sequence generated in this study showed considerable
variation with other species.
The BLAST analysis of 513 bp of the insect A. cerana showed
significant homology with A. cerana from Indonesia. The
phylogenetic NJ tree was carried out using MEGA6 software.
The NJ tree was constructed based on the multiple aligned
sequence data for different Apis species. The tree separates the
genomes into 3 main clades. All A. mellifera species were
included in one clade, A. cerana species in other clade and A.
koschevnikovi and A. florea in another clade.
The estimated transition/transversion bias (R) is 0.46. The
average nucleotide composition across the species is T=42.2%;
A=32.4%; C=15.3%; G=10.1%. These results show that
analysis based on mitochondrial gene can be useful for
unraveling phylogenetic relationships within the species A.
cerana. The percentage of A+T was higher than that of G+C
which reflected further in the codon usage. The second codon
position contained 70.9% of AT nucleotides and decreased to
59.7% in third codon position. AT nucleotide composition of
A. cerana from Kerala was higher than that of Indonesia
(Table 1). The branch length of A. cerana (Indonesia)
(GU191875) was less compared to A. cerana Kerala (GenBank
Accession No. KM230116), indicating less divergence from
its ancestor. The phylogeny analysis using NJ tree revealed the
sharing of common ancestor of these two species (Figure 1).
Fig 1: Phylogenetic tree of Apis cerana using NJ method

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Journal of Entomology and Zoology Studies
Table 1: Evolutionary divergence between COI sequences of A. cerana with other Apis species
Serial No. Species name with Accession Number % of divergence
1
Apis cerana
(Indonesia)(GU191875) 2.72
2
Apis cerana
(Japan) (AF153105) 6.04
3
Apis koschevnikovi
(AF153111) 6.53
4
Apis mellifera ligustica
(GU979498) 8.54
5
Apis mellifer
a carnica
(GU979496) 8.54
6
Apis mellifera adami
(AY114477) 8.54
7
Apis mellifera macedonica
(AY114473) 8.54
8
Apis mellifera caucasica
(AY114472) 8.54
9
Apis mellifera anatoliaca
(AY114471) 8.54
10
Apis mellifera sicula
(AY114483) 8.93
11
Apis melli
fera iberica
(AY114478) 9.38
12
Apis dorsata
(AF153113) 10.36
13
Apis florea
(AB284150) 11.97
4. Conclusion
Most of the phylogenetic studies are based on individual gene
or a few genes with similar evolutionary rate or entire
mitochondrial genome. DNA barcoding techniques have been
used to demarcate the phylogeographical variants. In
mitochondrial gene, the phylogeny and phylogeography of A.
cerana have been resolved. Geographically India is nearest to
Indonesia than Japan. So the high intraspecific nucleotide
distance observed is due to the geographical isolation of these
populations. The phylogenetically close species of A. cerana is
A. mellifera. This region has good discrimination power for A.
cerana. This study reveals that the former region is capable of
differentiating the phylogeographical variation of Apis species
found in world.
5. Acknowledgements
The financial assistance from University Grants Commission,
New Delhi and Kerala State Council for Science Technology
and Environment under Research Projects is gratefully
acknowledged.
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