Introduction
Honey is a natural product processed by honey bees when
they ingest nectar from flowers (Sajwani et al. 2007). The honey bee is
an important insect in Oman. Honey bees belong to the family Apidae (Hymenoptera) and they show complete metamorphosis (Yadav et al.
2017). Presently, there are ten species of honey bee recognised
belonging to the genus Apis,
including A. mellifera, A. cerana, A. koschevnikovi,
A. florea, A. andreniformis,
A. nulensis, A. dorsata,
A. binghami, A. nigrocincta
and A. laboriosa. Most of these species look
alike, with some differences in their colours and sizes. Three species of honey
bee are native to Asia and one species is native to the Euro-African region (Yadav et al.
2017). Based on sequence analysis, the genus Apis was found to cluster into
three sections: giant bees, cavity-nesting bees, and dwarf bees (Gupta 2014). Apis
mellifera has evolved into several subspecies,
which are grouped into four evolutionary branches: the European North
Mediterranean (C), the West-Mediterranean (M) lineages, the Oriental (O)
lineage and the African (A) lineage (Ruttner
1988; Garnery et al. 1992; Wallberg et al. 2014).
Two species of honey bees are known from Oman; the dwarf
honey bee (A. florea) which is locally known as “Abu Tuwaiq”
and the cavity-nesting bee (A. mellifera)
locally known as “domesticated Omani honey bee” (Al-Farsi et al. 2018). Apis florea mostly inhabits in mountains and trees. Bees
of this species build a single comb, and their honey is expensive. The
domesticated Omani honeybee (A. mellifera) was
imported from Yemen to Oman before 400 years by the King Saif
Bin Sultan Al-Yarubi. Omani beekeepers traditionally
use palm trunks to keep the honey of Omani honey bees. In the 1970s, the
Ministry of Agriculture and Fisheries of Oman started changing keeping honey
bees from palm trunks to Langstroth Beehive. A. mellifera produce higher amount of honey compared with A. florea.
In 2016, there were 100000 hives in Oman producing around 600 tons of honey
(Al-Farsi
et al. 2018).
Based on sequence data, the native honey bee in Saudi
Arabia is divided into three clusters (Alattal et al. 2014). The Ministry of
Agriculture and Fisheries of Oman did an experiment to compare the
morphological differences between Omani bees and other bees in the region (Elbassiouny 2009). They found that the length
of proboscis in Omani bees is different from Yamani, Carinolic
and Italian Bees. However, there are no studies related to the honey bee
population in Oman based on the sequence data.
Mitochondrial DNA (mtDNA) has
been widely used as a valuable tool in phylogenetic studies of species and
subspecies of honey bees (Garnery et al. 1992). Phylogenetic
analysis of honey bee populations is important as it helps identify the
relationship of honey bee populations from this part of the world to honey
populations in other countries. In addition, it helps identify the potential
presence of new genetic resources of honey bees for future studies that target
the improvement of honey quality and quantity.
Present study describe Omani honey bee populations using mtDNA COI–COII intergenic
region by comparing them to related honey bees in the Middle Eastern region.
Materials and Methods
Fifty
samples of honey bees were collected from 8 governorates in Oman (Al-Dhakhlia, Al-Batinah, Al-Dahira, Al-Sharqia, Al-Buraimi, Musandm, Al-Wusta and Dhofar) (Table 1). The
samples were kept in 50 mL tubes with 70% methanol and stored at -20°C.
All
the 50 samples were subjected to DNA extraction. However, each sample (one
honey bee) was grinded separately and used for DNA extraction according to Al-Sadi et al. (2012) to avoid contamination or mixing
two phylogenetic populations together. The mtDNA COI–COII intergenic region was amplified using the primer
pairs E2 (5’-GGCAGAATAAGTGCATTGGGC-3’) and H2 (5’-CAATATCATTGATGACCTTA-3’) (Cornuet et al.
1991; Garnery et al. 1992).
Polymerase chain reaction (PCR) was performed according to Garnery et al.
(1992) in an Applied Biosystems Veriti™ 96-Well Thermal Cycler using an illustra
PuReTaq Ready-To-Go PCR Beads. The denaturation was
at 92°C for 3 min, followed by 30 cycles of 92°C for 30 s, 47°C for 90 s, and
72°C for 45 s. This was followed by a final elongation step of 72°C for 10 min (Garnery et al.
1992; Syromyatnikov et al. 2018).
Sequencing
was conducted using the same primers used in PCR. The sequences from Oman were
compared with representative sequences of honey bees in GenBank
(National Centre for Biotechnology Information, NCBI). Sequences from this
study and refence sequences of 105 A. mellifera from GenBank were
aligned and optimized manually using MEGA v. 6 (Tamura et al. 2013). A maximum likelihood analysis was performed using raxmlGUI
version 1.3 (Silvestro
and Michalak 2012). The search for the optimal ML
tree was conducted with 1,000 separate runs and the bootstrap
support values above 50% were displayed on the tree. Apis mellifera ligustica (NC 001566) was used as an outgroup. Printing
of the resulting trees was done using MEGA v. 6, while Adobe Illustrator CS v.6
was used to prepare the layout.
Table 1: Sampling
details and their GenBank accession numbers
Sample No. |
Location |
DH2 |
Dalkoot |
S7 |
Al Kamel |
DH6 |
Takah |
DH3 |
Rkhioot |
S9 |
Al Kamel |
DH1 |
Dalkoot |
DH4 |
Rkhioot |
D5 |
Adm |
H8 |
Yankul |
H5 |
Dank |
S3 |
Wadi Bani Khalid |
H4 |
Ibri |
DH5 |
Rkhioot |
S8 |
Al Kamel |
H6 |
Ibri |
B10 |
Al-Auabi |
S1 |
Wadi Bani Khalid |
DH7 |
Takah |
B6 |
Al-Rustaq |
D6 |
Adm |
S5 |
Al Mudibi |
B3 |
Shinas |
D2 |
Izki |
D1 |
Izki |
H3 |
Ibri |
B4 |
Sohar |
B11 |
Al-Auabi |
B8 |
Al-Rustaq |
S6 |
Sur |
H7 |
Ibri |
B12 |
Al-Auabi |
H1 |
Ibri |
S10 |
Al Mudibi |
B7 |
Al-Rustaq |
S2 |
Wadi Bani Khalid |
BR1 |
Muhadah |
B9 |
Al-Rustaq |
B5 |
Al-Rustaq |
DH8 |
Murbat |
D3 |
Nizwa |
S4 |
Ibra |
B4 |
Sohar |
W1 |
Mohut |
D4 |
Al-Jable
Al-Akhdar |
M2 |
Khasab |
M1 |
Khasab |
H2 |
Ibri |
B4 |
Sohar |
D8 |
Al-Jable
Al-Akh |
DH10 |
Salalah |
Results
PCR
of the COI–COII intergenic
region produced three amplicon sizes: 612 bp, 812 bp and 1000bp,
designating for the populations PoQ, PoQQ and PoQQQ, respectively (Fig
1). The mtDNA COI–COII intergenic region of 50 honey-bee samples was
sequenced. The sequences of 29 representative samples were deposited in GenBank under the accession numbers from MF326653 to
MF326681. The majority of the samples in the present study belong to the PoQ sequences (52%) and PoQQ
sequences (42%). Only three samples (6%) were found to belong to PoQQQ and these samples were collected from Adam, Al-Jable Al-Akhdar and Dank.
Phylogenetic analysis was conducted based on the COI–COII sequence data of 50
individuals from the present study and a total of 105 members of Apis mellifera,
with Apis mellifera
ligustica (NC 001566) as the outgroup taxon. Phylogeny results indicated that a
haplotype of a single lineage was found in the samples collected from Oman
(Fig. 2). All these samples showed haplotypes known from the Oriental (O)
evolutionary lineage.
Discussion
Findings from this study show
that the Omani populations of honey bees show genetic differences and belong to
the Oriental (O) evolutionary lineage. Alattal et al. (2014) showed that most
honey bee populations from Saudi Arabia belonged to the O lineage, with only
few belongings to the A lineage. The individuals from Oman clustered
populations from Saudi Arabia, Yemen, Lebanon and other countries, which is
expected for Apis
mellifera from this part of the world. However,
the Omani populations showed subclustering within the
O lineage, which may indicates the presence of genetic diversity among A. mellifera population in the country (Techer et al.
2015).
Fig. 1: Structural organization of
the COI–COII intergenic region of mtDNA
on 1.5% agarose gel. L is
the 100 BP ladder; PoQ, PoQQ
and PoQQQ sequences correspond to the O lineage
Fig. 2: Maximum likelihood tree revealed by RAxML
from an analysis of COI-COII intergentic region
sequence data for Apis mellifera.
RAxML bootstrap supports (≥50%) are given at
the nodes and the tree is rooted to A. mellifera ligustica (NC 001566)
Conclusion
This is the first study that
characterized phylogenetic relationship of A. mellifera honey bees from Oman. It
indicates the presence of diversity in the population of these honey bees in
Oman. Future studies should address morphological features
of these honey bees and elucidate their genetic structure using population
genetic analysis.
Acknowledgments
Authors would like to acknowledge Mr.
Sultan Al-Harrasi, Mr. Haythem Al-Malki and Mr. Ibrahim Al-Hanai for
participating in the collection of samples and Beekeepers for help in the
collection of samples. Thanks to the Ministry of Agriculture and Fisheries and
Sultan Qaboos University for financial support of the
study.
Author
Contributions
KH Al-Aghbari, HM Al-Sabbari and KS
Al-Maani collected samples, extracted DNA and did PCR, SS Maharachchikumbura
and AM Al-Sadi did phylogenetic analysis, all authors wrote and approved the
manuscript
References
Alattal Y, M
Alsharhi, A Alghamdi, S Alfaify, H Migdadi, M Ansari (2014). Characterization
of the native honey bee subspecies in Saudi Arabia using the mtDNA COI–COII
intergenic region and morphometric characteristics. Bull Insectol 67:31‒37
Al-Farsi M, S
Al-Belushi, A Al-Amri, A Al-Hadhrami, M Al-Rusheidi, A Al-Alawi (2018). Quality
evaluation of Omani honey. Food Chem
262:162‒167
Al-Sadi AM, AG
Al-Ghaithi, ZM Al-Balushi, AH Al-Jabri (2012). Analysis of diversity in Pythium
aphanidermatum populations from a single greenhouse reveals phenotypic and
genotypic changes over 2006 to 2011. Plant
Dis 96:852‒858
Cornuet JM, L Garnery,
M Solignac (1991). Putative origin and function of the intergenic region
between COI and COII of Apis mellifera L. mitochondrial DNA. Genetics 128:393‒403
Elbassiouny A (2009). Mean
Morphological Measurements Omani Bees Compared to the Yemeni Bees and the
Cranolic and Italian Bees, p:3. MAF (Eds.). Muscat, Oman
Garnery L, JM Cornuet,
M Solignac (1992). Evolutionary history of the honey bee Apis mellifera
inferred from mitochondrial DNA analysis. Mol
Ecol 1:145‒154
Gupta RK (2014).
Taxonomy and distribution of different honeybee species. In: Beekeeping for
Poverty Alleviation and Livelihood Security, pp:63-103. Springer, Cham,
Switzerland
Ruttner F (1988). Biogeography and Taxonomy of Honeybees, Springer-Verlag,
Berlin, Germany
Sajwani A, SA Farooq,
A Patzelt, EA Eltayeb, VM Bryant (2007). Melissopalynological studies from
Oman. Palynology 31:63‒79
Silvestro D, I
Michalak (2012). RaxmlGUI: A graphical front-end for RAxML. Org Divers Evol 12:335‒337
Syromyatnikov MY, AV
Borodachev, AV Kokina, VN Popov (2018). A molecular method for the
identification of honey bee subspecies used by beekeepers in Russia. Insects 9:10-21
Tamura K, G Stecher, D
Peterson, A Filipski, S Kumar (2013). MEGA6: Molecular evolutionary genetics
analysis version 6.0. Mol Biol Evol
30:2725‒2729
Techer MA, J
Clémencet, P Turpin, N Volbert, B Reynaud, H Delatte (2015). Genetic
characterization of the honeybee (Apis
mellifera) population of Rodrigues Island, based on microsatellite and
mitochondrial DNA. Apidologie 46:445‒454
Wallberg A, F Han, G
Wellhagen, B Dahle, M Kawata, N Haddad, ZLP Simões, MH Allsopp, I Kandemir, PDL
Rúa, CW Pirk, MT Webster (2014). A worldwide survey of genome sequence
variation provides insight into the evolutionary history of the honeybee Apis mellifera. Nat Genet 46:1081‒1088
Yadav S, Y Kumar, BL Jat (2017). Honeybee:
Diversity, castes and life cycle. In: Industrial Entomology, pp:5‒34.
Springer, Cham, Switzerland