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

 

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