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The European honeybee, Apis mellifera L. plays an important role in pollinating flowering plants and in conserving and promoting biodiversity and agricultural productivity (Klein et al. 2007). One third of the total human diet is dependent on insect-pollinated plants, predominately by the European honeybee (McGregor 1976). A number of crops either depend mainly on or are benefited by honeybee pollination. In North America, for example, honeybees pollinate no fewer than 90 crops (Partap 2011) (In California, almond production is a USA$2 billion dollar industry, and is almost entirely dependent upon honeybee pollination (Council 2007). The monetary value of honeybee pollination is estimated at about USA$15-20 billion dollars annually (Johnson 2010).
Beekeeping is one of the longstanding traditional and environmentally friendly agricultural activities in Saudi Arabia. It is contributing significantly for economic and social development of the country through providing alternative or additional income and livelihoods of communities through the sale of honey and enhancing agricultural productivity, conserving environment and biodiversity. However package bee importations, of more than 100,000 colonies each year, may promote successful pollination and conservation of plant flora in the kingdom, it may increase an unwanted hybridization of the local bee race with other bee races.
To increase honey production, beekeepers in Saudi Arabia import more than 100,000 Colony every year. However, more than 70% of these colonies die out every year due to abiotic stresses, primarily due to high air temperatures (Al Ghamdi 2005; Alqarni 2006). The native honeybees could become endangered due to annual introduction of honeybees from different sources, genetic dilution, erosion and it is predicted that within a few years, leading to the absence of pure genetic resources will from the native bee. In this regards, the degree of genetic dilution increases and currently, it is difficult to determine where pure native honeybees could be found in the Kingdom of Saudi Arabia. In addition, a few studies were done regarding to biological, morphometric, genetic variability, and identity of native bee races in Saudi Arabia (Alqarni 1995; Alqarni 2006; Alqarni 2011;under investigation 2011).
The native honeybee race is the most precious genetic resource. The estimated number of honeybee colonies in Saudi Arabia is between 700,000 to 800,000 owned by approximately 4,000 beekeepers. Approximately, 200,000 of the above number are the native subspecies A. m. jemenitica (Alqarni 2011).
The only native subspecies of the honeybee, A. m. jemenitica occurring in Saudi Arabia, is uniquely adapted to survive to the hot air temperature conditions (Nikolenko and Poskryakov 2002). Therefore, it is very important to conserve and improve the native honeybee through selection, breeding, and conservation program of native honeybee populations. This will have a great impact on improving the beekeeping industry of Saudi Arabia. It is difficult to conserve and improve the native honeybee subspecies without identification and assessment of its degree of genetic integrity through analysis of morphometeric and genetic characters. This identification and assessment is an essential step in conserving and improving programs of the native honeybee, A. m. jemenitica. Research is part of a project Funded by The National Plan for Science and Technology.
Assess the genetic purity of native honeybee, A. m. jemenitica using mitochondrial DNA molecular markers and nuclear microsatellites techniques.
Identification of morpometric markers of honeybee races including the native races correlated to the genetic analysis in Saudi Arabia
Assessing the extent of gene introgression of introduced honeybees with A. m. jemenitica
Localizing and positioning pure A. m. jemenitica distributions within Saudi Arabia.
Many morphometrical characters are used for a long time to differentiate between honeybee races and populations Morphometrics is the measurement and analysis of morphological characters and widely applied to study insect life history, physiology and systematic. Also Morphometric measures both genotypic and phenotypic characteristics. These include three categories: pigmentation, body size, and venation of forewing angle, which represent 82.1% of the total variation found in A. mellifera. The body size factor is negatively correlated to temperature and positively correlated to altitude and represents about 45.5% of the variation. Pigmentation of the tergite represent about 18.6% of the variation, which is associated with temperature, altitude and rainfall (Amssalu et al., 2004). Morphometric analysis has been used as a useful tool to assess subspecies limits in A. mellifera (Ruttner and Louveaux 1978; Ruttner 1988), and to study of genetic variability of honeybees (Garnery et al., 1992; KekeçoÄŸlu et al., 2007; Miguel et al., 2010).
Historically, the classifications of the honeybees of the world, thirty-six characters were identified. By applying multivariate and stepwise discriminate analysis techniques, these characters were reduced to ten by (Ruttner and Louveaux 1978), 13 by, Amssalu et al., (2004) or 11 characters by Andere et al. (2008) for separation of honeybees of different geographical origin in Africa. Based on morphometric characters, the honeybee, A. mellifera were classified into about 25 recognized subspecies throughout the world Ruttner 1988 . These subspecies were grouped into five major evolutionary lineages as follows: the A (Africa), M (Western Europe), C (South-Eastern Europe), and O (Middle East), and Y (Yemenitica) lineage (Ruttner and Louveaux 1978; Ruttner 1988; Sheppard et al., 1997; Franck et al., 2001). This classification was largely supported by mitochondrial DNA studies (Garnery et al., 1992; Arias and Sheppard 1996; Franck et al., 2000), and microsatellite loci data (Miguel et al., 2010).
Characterization of native honeybee
Native honeybee race in Saudi Arabia is belongs to linage Y (Franck et al., 2001). The first record of honeybee in Saudi Arabia was described by Ruttner (1976) as a new subspecies; A. m. jemenitica, specimens collected from only six locations within the Kingdom: two samples from Jazan, two samples from Riyadh and two samples from Alhasa (Ruttner 1988). The same subspecies was also found in Yemen (Ruttner and Louveaux 1978), Sudan (Ruttner and Louveaux 1978; Rashad and El-Sarrag 1980; El Sarrag et al., 1992), Chad and Oman (Engel 1999), Somalia (Ruttner 1988), and Ethiopia (Hepburn and Radloff 1998; Amssalu et al., 2004). However, the smallest Yemeni bee was recorded in Saudi Arabia (Ruttner 1988). It covers the western, southern and southeastern parts of the Arabian Peninsula where the A. m. jemenitica adapted to environment (Karpowicz 1989 ).
Apis m. jemenitica is a small bee that occuring in hot arid zones of eastern Africa and the Arabian Peninsula (Aqlan 1999; Engel 1999). The morphometrics of A. m. jemenitica are placed in the lowest sector of the total variability, overlapping those of A. cerana (Ruttner 1976) .The morphometric variation in Yemeni bees may be due to congenital aspects, environment, latitude and altitude. Also recorded in Sudan subspecies corresponds very closely to A. m. jemenitica as described as small body size, slender and very yellow (Rashad and El-Sarrag 1980). According to morphometric analysis two differentiated populations of A. m. jemenitica were described from Yemen, but it belongs to the same subspecies However, the population of Aden was smaller in size than other populations of A. m. jemenitica and deviated by 6.24-19.06% from those recorded in many references of the same subspecies (Khanbash 1990).
Because of limitation of morphometric analysis in measurement of genetic diversity, a number of markers as fingerprinting methods have been development for the determination of genotype which are not susceptible to environment effects such as: allozymic variation (Contel et al., 1977). random amplified polymorphic DNA (RAPD) (Williams et al., 1990), amplified fragment length polymorphism (AFLP). Besides, whole genome microarray and single nucleotide polymorphism (SNP) (Whitfield et al., 2006). . These markers have been contributed significantly to analysis and understanding genetic diversity (Behura 2006) and also become a popular means for identification and characterization of plant and animal species (Shaw et al., 2002).
Mitochondrial DNA (mtDNA) Marker
The (mtDNA) is a closed-circular, double-stranded molecule, ranging in size from 15 to 20 kb (Wolstenholme 1992). Analysis of mtDNA variations have been used to differentiate between five lineages of honeybees (Garnery et al., 1992; Franck et al., 2000; Palmer et al., 2000; Sheppard and Smith 2000). Mitochondrial sequences are known to be useful in discriminating among honeybee lineages and variation between and within honeybee population (Cornuet et al., 1991; Smith et al., 1991; Garnery et al., 1992; Moritz 1995). All honeybees have a non-coding sequence, which appears to be unique to bee genus Apis sp, located between the mitochondrial COI and COII genes (Cornuet et al., 1991). In the honeybee, as in most animals, mitochondria are inherited only from the mother, so all individuals in a colony share the same mitochondrial DNA (Crozier and Crozier 1993). It reveals more than 50 haplotypes in lineages A and M (Garnery et al., 1992) but only six haplotypes in lineage C (Franck et al., 2001; Kandemir et al., 2006; Ozdil et al., 2009).
Restriction fragment length polymorphisms (RFLPs) using mtDNA has been useful marker for the study of geographic origin and genetic variation in honeybees (Hall 1990; Hall 1991; Arias and Sheppard 1996; Alippi et al., 2002). The most widely used mtDNA marker gave variation was in the intergenic region between the COI and the COII gene in Apis mellifera (Garnery et al., 1992). Mitochondrial COI-COII intergenic region of the honeybee have been useful in differentiating evolutionary lineages and groups of subspecies (Cornuet et al., 1991; Hall 1991; Garnery et al., 1992; Franck et al., 2000). So the analysis of mitochondrial COI-COII will be essential to differentiate between the native honeybee and other honeybees subspecies.
Simple sequence repeated (SSR) or microsatellites markers
SSR markers are tandem repeats interspersed throughout the genome and they can be amplified using primers. SSR has been successfully used to study genetic variation within populations of the same species, such as honeybees (Brown et al., 1996). This marker maternally and paternally inherited, which enables hybridization and gene introgression studies, allowing the distinction between homozygous and heterozygous individuals, and commonly used to distinguish honeybee lineages on the basis of morphometric (Ruttner 1988) and mtDNA (Smith et al., 1991), and subspecies (Hall 1986;1990; Hall and Smith 1991). Microsatellite markers from single and multiple loci specific to eastern European, western European, and African bees have been used to characterize Old World European and African honeybee populations (Estoup et al., 1993; Estoup et al., 1995).
In the present investigation, mtDNA markers and SSR markers combination will be used to study the genetic diversity of honeybees from localities in Saudi Arabia. Additionally, it will be analyze morphometrically variable populations for genetic structure. This aspect of the study will focus on non-migratory colonies along Sarawat Mountains and Tehama Plains sites that have been kept isolated from foreign queens introductions, so that it may well reflect the ancestral genetic characteristics of honeybees localities in western and southwestern Saudi Arabia. In addotion, this study will assess the purity of Saudi non-migratory colonies of A. m. jemenitica.
Materials and Methods
Initial surveys will locate native honeybee populations in the Kingdom of Saudi Arabia, focusing on current distribution of A. m. jemenitica. The study will cover all regions of Saudi Arabia. Representative samples will be collected from all regions especially from areas of active beekeeping. Stratified sampling of three levels; region, province and locations will be used (Bee et al., 2010).
Identification of regions, provinces and locations
The Saudi Arabia divided geographically into thirteen regions according to the current official administrative division. Samples will be collected from each region according to stratified Sampling Technique. So the samples will be classified to three strata: high density, medium density, and low density populations according to the number of native honeybee colonies in Saudi Arabia or in the region (Alqarni 2011). The Quota of collected samples will be 42% high density areas, 44% middle, and 14% small of the total samples. The Quota will be distributed equally for all of the regions. The Quota of each of the regions will be 21% for high density, 11% medium and small 2% of the total samples (Appendix 1).
It will be interview with beekeepers in every region of country to obtain localities of active apiaries within the provinces. Subsequently, it will concentrate on region where isolated non-migratory bee colonies (A. m. jemenitica) occur and areas with imported honeybees that have existed on the same site for at least four years or moved within the region for more than 100 km in any direction. In additional samples will be collected from imported bees to assess potential gene flow between A. m. jemenitica and imported honeybee colonies. Sample size
The number of honeybee colonies in Saudi Arabia are estimated between 700,000 to 800,000 owned by approximately 4,000 beekeepers. Approximately, 200,000 of the above number are the native subspecies A. m. jemenitica (Alqarni investigation 2011). The representative sample of each population will be about 200 sample, which represents approximately. 0. 1% of the total number of native honeybee colonies as shown in Appendix (1).
Sample collection Method
About 100 adult worker bees will be randomly collected from each hive. After that, samples will be divided into two parts; the first part will be used for morphometric analysis and the second part for genetic analysis. The first part will be treated by hot water (to obtain fully stretched proboscis of the dead bees), put it into small plastic bottles, preserved in 70% ethanol with glycerin and be labeled (Ruttner and Louveaux 1978). The second part will be immediately preserved in eppendorf tube by immersion absolute ethanol, Change the ethanol after 12-l6 h , then stored at -20 Â°C during the collection and then transported to the laboratory to be stored at Â-80Â°C until used for DNA extraction (Hall 1995).
Localizing and positioning
During the collecting, a GPS unit will be used to determine elevation; map coordinates for each hive of native honeybees and converted to a layer of GIS. Program Map will be used and survey to determine locations of honeybee hives. Also, all necessary environmental information will be recorded and if imported honeybee colonies are nearby.
Ten workers from each colony will be dissected under binocular microscope and mounted as follows: dehydrated in alcohols, cleand in xylol, place on slides and fixed using canada balsam (Adsavakulchai et al., 1999). The morphometric characters will be processed using the computer software; (UTHSCSA Image Tool software, University of Texas Health Science Center San Antonio Image Tool Ver. 3.00) under resolution up to 2400 dpi. (Wilcox et al., 2002).
For each individual bee, twenty morphometric characters that have high discriminatory power will be measured following (Ruttner 1988). These characters include: Length of proboscis (5). Length of femur (Fe) (6), Length of tibia (Ti) (7), Length of tarsus (Ta) (8), Longitudinal diameter of tergite 3 (T3) (13), Longitudinal diameter of tergite 4 (T4) (14), Longitudinal diameter of sternite 3 (15), Longitudinal of mirror sternite 3 (16), mirror transversal sternite 3 (17), Distance between mirror (18), Longitudinal dimeter of sternite 6 (19), Transversal of sternite 6 (20), wing length(21), wing width(22), Number of hamuli(42), Cubital index a/b (27\28), wing angle B4 (22), wing angle N23 (30), wing angle O26 (31), Pigmentation of abdominal tergite 2 (32), pigmentation of abdominal tergite 3 (33), pigmentation of abdominal tergite 4 (34).
Reference data of honeybee subspecies will be obtained from the database of the Institut für Bienenkunde, Oberursel in Frankfurt, Germany. Which include A. m. jemenitica, A. m. carnica, A. m. syriaca, A. m. ligustica and. A.m meda (.
Data will be subjected to principal component analysis and discriminate analysis using the SPSS statistical software. (Kandemir et al., 2006).
Total nDNA will be extracted from the thorax of a single adult worker per colony using a DNeasy Blood & Tissue Kit (Qiagen, Valencia, California). The same sample will be used for both mtDNA and microsatellite loci analyses. The protocol followed will be according to the manufacturer's instructions for animal tissue (Appendix 2).
PCR amplifications (mtDNA analysis)
mtDNA analysis will be performed on the intergenic region between the COI and COII genes. This region will be amplified using DNA from only one bee worker per colony by using PCR and primers E2 5' -GGCAGAATAAGTGCATTG - 3', H2 5'- CAATATCATTGATGACC-3' (Cornuet et al., 1991; Garnery et al., 1992). Each sample reaction will be performed in 25 Î¼L containing 2.7 Î¼L of Taq buffer (Promega, Madison, WI), 1.5 mM MgCl2, 25 nmol of each dNTP, 25 pmol of E2 and H2 primers, 11.8 Î¼L of distilled water, 0.5 Î¼l (5 U/Î¼l) of Taq polymerase (Promega), and 1Î¼L of DNA extract. The PCR conditions for all reactions will be one 2 min cycle at 95 °C, 30 cycles of 95 °C for 30 s, 54 °C for 30 s, 72 °C for 30 s and a 5 min cycle at 72 °C in a GeneAmp 9700 thermocycler (Applied Biosystems). To determine the size of the amplified region, a 6 Î¼L aliquot of PCR product electrophoreses on a 1.4% agarose gel. And visualize with ethidium bromide in UV light with aÂ Gel DocÂ imager (syngene).
Digestion with restriction enzymes
5Î¼L from PCR products from each sample will be digested for 3 h at 37 CÂ° with 0.2 Î¼l of BSA (Promega), 2 Î¼l of enzyme buffer (Promega) and 1 Î¼l of restriction enzyme (Promega); the final volume will be adjusted to 20 Î¼l by adding sterile distilled water (Shahera Zaitoun et al., 2008). The restricted DNA fragments will be separated on a 10% polyacrylamide gel or 2% Metaphor agarose and stained with ethidium bromide. The restriction fragment will be scored as present (PCR product cut) or absent (PCR product not cut), based on the visualization of a two-band pattern or one-band pattern on the gel according to (Garnery et al., 1998).
Colonies will be classified according to their mtDNA haplotype. Each new haplotype sequenced to confirm its uniqueness and identity with haplotypes published by (Franck et al. 1998). The PCR products will be purified using QIAquick Gel Extraction Kit (QIAGEN). Sequences of haplotypes will be aligned with published sequences of 44 different haplotypes of honey bees manually with Cluster software (Thompson 1999).
Polymerase chain reaction (PCR) will be amplified eight specific microsatellite loci by Eight pairs specific primers namely (A7, A24, A28, A88, A113, B124 (Estoup et al., 1995), Ap43 (Garnery et al., 1998), Ap81 (Solignac et al., 2003). These loci have been shown to be good proxies for assessing genetic variation in honeybees at the population level.
PCR will be amplified in 20 ÂµL reactions containing 1 ÂµL extracted DNA, 1Ã- reaction buffer Promega (Madison, WI), 3 mM dNTP mixture, 1.0-4.0 mM primer, , 1.5 units Taq polymerase (Promega, Madison WI) and the MgCl2 1.5 mM . The uniform PCR will be used for all reactions : one cycle 2 min at 95 °C, 30 cycles of 95 °C for 30 s, 54 °C for 30 s, 72 °C for 30 s and a 5 min cycle at 72 °C. PCR products will be visualized on 2% Metaphor agarose stained with ethidium bromide. Microsatellite fragment sizes will be scored by comparing the length of the PCR fragments to the standerd 100 bp (Qiagen).
For Mitochondrial analysis unbiased estimates and standard deviations of gene diversity of mtDNA (Nei and Tajima 1981) and estimations of the population subdivision will be documented using the Arlequin software package (Schneider et al., 1997).
Microsatellite allele sizes will be scored by comparison of the lengths of the PCR products to the stander 100 pb marker (qiagem). The number of alleles per locus (n) and expected heterozygozity (He) will be estimated with GENEPOP v3.4 software (Nei 1973; Raymond and Rousset 1995). Hardy-Weinberg test will be performed by the Markov chain method for every locus in each locality (Guo and Thompson 1992). The deferent apiaries will be tested for population differentiation with the Fisher exact test (Raymond and Rousset 1995). The cyto-nuclear disequilibrium will be analyzed with the same test (Asmussen et al., 1987).
Research Time Line
Type of work
preparing and ordering of materials and equipments
Surveying and sample collection
Total DNA extraction and purity and concentration of DNA determined
PCR amplification for RFLP
Type of work
PCR amplification for Microsatellite
writing and publication