EurBee board Dorothea Brückner, Germany Norberto Milani, Italy Robert Paxton, Great Britain Dalibor Titěra, Czech Republic Bernard Vaissiere, France Program consultant

Diversity and conservation

Area de Biología Animal, Fac. Veterinaria, Universidad de Murcia (Spain), E-mail: pdelarua©um.es

The mating frequency of two unmanaged populations of Apis mellifera iberiensis, one located at the north of the Iberian Peninsula corresponding to the mitochondrial West-European (M) evolutionary lineage, and other one at the southeast bearing African (A) mitochondrial DNA, has been estimated using five microsatellite loci. A total of 1441 workers were genotyped and the estimated, effective and observed numbers of matings were calculated from the workers offspring. The queens of the twenty-two M colonies analysed mated 8-21 times with an average relatedness among workers of 0.303±0.020. The observed number of matings per queen in the twelve A colonies ranged from 12 to 25 and the genetic relatedness among these workers was 0.294±0.102. These results are within the range of those found in other Apis mellifera subspecies. A significant difference (p=0.0548) has been detected between the two types of honeybee populations in relation to the observed number of matings (15.7±4.6 averaged for those colonies corresponding to the M lineage and 18.9±6.5 for the colonies belonging to the African lineage). These results suggest that ecological factors as the location of the colonies and climate conditions as well as evolutionary trends may influence the queen´s mating behaviour.


Introgressions and structure of the genetic diversity of the black honeybee in France

A. Rortais, G. Arnold, L. Garnery

CNRS, E-mail: rortais©pge.cnrs-gif.fr

The objective of this study is to assess the situation of the black honeybee in France. A large survey has been conducted over the past three years (2003-2006) to determine the levels of introgressions and genetic diversity of honeybees in various regions of France and in South of Belgium. A total of about 3800 colonies were sampled and analysed (mtDNA for all colonies and microsatellites for a few populations). Fifty-one new haplotypes were discovered in France, doubling the total amount of haplotypes known in the M lineage. These results highlight the high diversity found in populations of the black honeybee. This diversity is geographically highly structured. At a large scale, it exhibits a sharp gradient from south-west to North-East of France with predominance of haplotypes M4 and M19 in the south and M17 in the north. At a finer scale, local differentiations appear that need further investigation to determine if they are associated to local differenciations. Further discussion is given on the structure of the diversity found in France compared to other European countries where A. m. mellifera naturally occurs.


Discrimination between honeybee subspecies based on geometric morphometrics

Adam Tofilski

Department of Pomology and Apiculture, Agricultural University of Krakow, E-mail: rotofils©cyf-kr.edu.pl

Discrimination between honeybee (Apis mellifera) subspecies is usually performed by methods of traditional morphometrics, which is based on multivariate analysis of distances, angles and ratios. Those methods proved to be successful, however, there are some difficulties related to interpretation and graphical presentation of results. Many of the problems of traditional morphometrics were solved by geometric morphometric. The new method is based on landmarks, which are described by Cartesian coordinates.

The methods of geometric morphometrics were used to analyse differences between three honeybee subspecies: A. m. mellifera, A. m. carnica and A. m. caucasica. From every subspecies a hundred workers were collected and their forewings were dissected. As landmarks 18 vein junctions were chosen. After obtaining the wings images the vein junctions were detected automatically using DrawWing software. Generalized Procrustes analysis followed by Canonical Variates Analysis was used to compare the shape of venation. The discrimination based on the geometric morphometrics proved to be successful. The analysis revealed many differences between the subspecies, which are difficult to detect using other methods.

E-mail of the corresponding author: icakmak©uludag.edu.tr

The goal of this study is to determine the variation of honey bee populations and also whether migratory beekeeping or importation of commercially produced queens would influence the natural distribution.

Honeybee samples were collected during May-August of 2002-2005 in northern Turkey, including South Marmara and Thrace along the coast from the Georgian to the Bulgarian border. A total of 58 colonies were sampled ranging from sea level up to 3000 m. Bee samples were taken from remote villages with no or minor migratory beekeeping activities within short distances and preserved in 70% ETOH until analysis. 38 morphometric characters (Ruttner 1988) were measured by using a stereo microscope and a PC-based video measuring system (Meixner 1994). The data were analyzed with the SPSS using factor analysis and discriminant analysis. Reference samples from the Oberursel data bank were included in the analysis.

The results of the morphometric analysis suggest that A.m. anatoliaca is the predominant race in Northern Turkey extending into Thrace region. A. m. caucasica is prevalent only in some Eastern Black Sea locations. Even though the bee samples were taken from stationary beekeepers and native racial distribution is predominantly preserved, some mixing was apparent throughout the region.


GENETIC DIVERSITY OF BEE ECOTYPES IN TURKEY-GREECE BORDER LINE USING MORPHOMETRIC AND MOLECULAR MARKERS

M. Kekecoglu ¹, M. Bouga ², P. Harizanis²,M.I. Soysal<sup>1

1</sup> Faculty of Agriculture, Department of Animal Science, Trakya University, Tekirdag, Turkey

² Laboratory of Sericulture & Apiculture, Agricultural University of Athens, Athens, Greece
E-mail: meralbilgilisoy©yahoo.com

According Ruttner’s morphometrics analysis (1988) A.m. syriaca, A.m. meda, A.m. anatoliaca and A.m. caucasica occur in Turkey, as well as, A.m. macedonica, A.m. adami, A.m. cecropia and A.m.carnica in Greece.In this study honey bees from 36 colonies from 4 different areas of Turkey-Greece border line were surveyed with morphometrics analysis using twelve characters and molecular analysis using PCR-RFLP’s method, on two mtDNA gene segments. The results of the morphometrics analysis were statistically processed using NTSYS program package; as concerning the results from mtDNA analysis REAP and PHYLIP packages were being applied.

The results of our research from both approaches show that populations studied are no the same. Diagnostic patterns have been revealed discriminating Greek and Turkish honey bees concerning mtDNA analysis.

The above mentioned results could be useful for conservation purposes concerning local honey bee populations.

mbouga©aua.gr

Since August 2005, the Laboratory of Agricultural Zoology and Entomology of AUA and the Hellenic Institute of Apiculture of NAGREF, have started an attempt to establish the National Certification System of Honey bee Queens in Greece. According to the above system, the Hellenic Accreditation System following the procedures of ELOT EN 45011 will accredit two bodies for Certification. The two bodies involved will be independently accredited, but they will collaborate very closely, also with other accredited laboratories. The Laboratory of Agricultural Zoology and Entomology will handle all procedures concerning the genetic origin of the queens produced in Greece and the Hellenic Institute of Apiculture will measure several parameters affecting the quality of the queens. The above system is co-financed by the EU and the Greek Government for 2006 and 2007.

Dipartimento S.En.Fi. Mi.Zo. Università degli Studi di Palermo, Italy; Istituto Zooprofilattico Sperimentale della Sicilia, E-mail: Sinacori©unipa.it

The genus Apis includes species mainly located in Asia. The unique species located in Africa, Europe and western Asia is Apis mellifera L.. In Italy this species includes 2 subspecies A.m.ligustica Spinola and A. m. sicula Montagano. This strain is present essentially in Sicily and because of less cross breeding selection original wild characters are probably well preserved. Biochemical characterization is classically used as tool to identify the different strains. However to aim the biodiversity preservation, in the last few years several studies had been started to identify molecular markers that can distinguish among different subspecies. Molecular approach can be useful to improve the classification systems and to discover more informative polymorphic sites. In this paper preliminary data on genetic polymorphism among the two different subspecies A. m. ligustica and A.m.sicula by analysis with different molecular approach (restriction analysis in mitochondrial DNA, AFLP, microsatellites), identified by iso-enzymatic pattern, are reported.


Hybridization among European subspecies of the Western honeybee Apis mellifera

Gabriele Soland

University of Bern, E-mail: gabriele.soland©honigbiene.ch

Modern Beekeeping practice has lead to many introduction of European honeybees into formerly bee free areas and areas preoccupied by other subspecies. The sudden sympatric occurrence has lead to the hybridization of subspecies of the Western honeybee Apis mellifera and population amalgamation would be expected to be the long term results. Especially subspecies with no or very few remaining allopatric populations are feared to be highly threatened by hybridization. The example of the Africanized honeybee has shown that complete admixture is not necessarily occurring if different honeybee subspecies are mingled. Many scenarios have been proposed in order to explain the unidirectional and non-random introgression proportions among European and African subspecies. Among European subspecies, many studies have shown, that hybridization does occur, but so far it is unknown to what extend hybridization leads to genetic introgression. In this study we quantify genetic differentiation and admixture proportions between the endangered A.m.mellifera and south-eastern European subspecies in populations with different levels of artificial selection. We show that there still is a high level of differentiation among the different subspecies. Although fertile hybrids are produced, admixture patterns show that there is a barrier to hybridization that prevents subspecies from complete admixture. This barrier can not be explained by artificial selection alone, what leaves the implication, that there exists a mechanism that hinders hybridization to some extend.


Microsatellite analysis revealed the maintenance of genetic identity of subspecies of Apis mellifera L. in Turkey in spite of intensive migratory beekeeping

C. Bodur, M.Kence, A. Kence

Middle East Technical University, Department of Biology

06531 Ankara Turkey

E-mail of the corresponding Author:aykut©metu.edu.tr

Despite the intense migratory beekeeping within the last three decades, a very high allozyme diversity have been observed between the honeybee populations distributed throughout Turkey. Our aim was to assess the present genetic structuring in the honeybee populations of Turkey using nine microsatellite loci, since it is claimed that gene pools belonging to different subspecies lost their integrity because of hybridization due to intensive migratory beekeeping. Honeybee populations were sampled from 11 provinces in Turkey and an additional sampling was carried out from Cyprus.

We have observed great genetic diversity among populations as indicated by significant pairwise FST values for the majority of population pairs, 52 out of 66 pairs. Also the most of the Nm values (pairwise number of migrants) were lower than 2 which indicates significant reduction of gene flow between these populations. The percentages of correct assignments of individuals to the population they were sampled, revealed a highest score as 87% and a 62% mean correct assignment score with a great variance because of the 3 populations that had very low assignment percentages. These populations were on the major routes of migratory beekeepers. The strategies for conservation of genetic diversity of well-adapted local honeybee populations in Turkey are being developed.

Voronezh State Agricultural University, ul. Michurina,1, Voronezh, 394087, Russia; E-mail: dobrynin©vsau.ru

The essence of ecologization of entomophilic crop cultivation is to bring insect-pollinated crop ecosystem function near to natural ecosystem function, i.e. to imitate natural processes, based on a balance basis, conservation and reproduction of wild life resources (Dobrynin, 1998, 2004). Native wild bee species are classified as valuable natural resources as well. Accordingly, the solution of the problem of sufficient supply of entomophilic crops with adequate pollinators should be based first on the active utilization of native widely spread bee species. There was developed the system of measures on conservation and increase the native pollinator numbers in natural habitat ((Dobrynin, 1998).

This system consists of the following components:

1. Conservation and creation of native bee nesting habitats

which include: protection of existing (creation of microrefuges) and enlargement of natural nesting habitats; establishment of new habitats favorable for pollinators nesting (imitation of natural nesting habitat); ecologization of techniques of establishment and management of entomophilic crop agroecosystems.

2. Enlargement and management of forage resources of native bees

which include: imitation of natural change of flowering vegetation in limits

of agroecosystems (using of bait crops, flowering adjustment and rational utilization); enlargement of entomophilic crop agroecosystems.

3. Pollinator protection in a system of chemical crop protection

taking into consideration: ecological features of pesticide-pollinator relationships (the role of abiotic and biotic factors in pesticide–pollinator relationships, influence of pesticide properties, regulations and objects of application on pollinators); special features of entomophilic crop protection; protection of pollinators during pesticide application.

Flowering set-asides in large cultivated zones: converging interests for farmers, the biodiversity, landscapes and beekeeping

Axel Decourtye

ACTA, E-mail: axel.decourtye©acta.asso.fr

Recently a decline in the bee populations has been observed in Europe. This assessment raises the question of the possible risk of the disappearance of all pollinating insects and the repercussions on human activities based on the survival of these insects (beekeeping, fruit, vegetable, and seed production). A reason for this decline will be the destruction and the fragmentation of the bees’ habitat. The preservation of their habitat is necessary but has never been taken into account. Fallowing area initiated by the European common agricultural policy might be an opportunity for apiculture and more generally for insect pollinators conservation. Appropriately sown, these agricultural surfaces could offer various sources of nectar and pollen. Agro-environmental set-asides might have positive impacts on insect populations by giving them habitat and food. European regulations impose fallowing area to be spontaneously or artificially sown. Allowed species are recorded in a list which can be modified on a regional scale. In France, specific clauses have been generated from the general frame in order to respond to particular situations: set-asides intended to preserve biodiversity and protect water quality. Evolution of rules and regulations as well as agricultural practices is necessary in order to improve insect population development. Sowing fallowing area with melliferous and polleniferous species would be a good contribution to promote beekeeping and protect biodiversity.
Determination of Genetic variation in Northern Iran honeybee (Apis mellifera meda) populations using microsatellite and RAPD markers

M.Kence, H. Jabbarifarhoud, R. Ivgin Tunca

Department of Biology, Middle East Technical University

06531 Ankara, Turkey

E-mail of the corresponding Author:mkence©metu.edu.tr

The main objective of the present study was to analyze the genetic structure of four honeybee populations (Sarein, Amol, Urmiye and Viladereg) from northern Iran using five microsatellite loci and RAPD primers. Average heterozygosity was estimated between 0.583 and 0.730 based on microsatellite data. Heterozygosity values for Iranian populations were found higher than European populations (0.230-0.395) but lower than the tropical African populations (0.756-0.896). Genetic differentiation among populations was estimated by using fixation index (FST) ranging from 0.032 to 0.142. Linkage disequilibrium tests for all pairs of loci at all populations detected 8 significant values between pairs of loci. The gene flow (Nm) between Iran populations was estimated as 1.570. Hence there is significant genetic differentiation among populations. A phylogenetic tree was constructed based on genetic distances among populations and clustered the Urmia-Viladereg into one and Amol-Sarein into another cluster.

On the other hand, genotype assignment tests based on the RAPD data analyzed by AFLPOP wich does the assignment of the likelihoods of each individual for different population pairs revealed distinction between Amol-Viladereg and Sarein-Viladereg population pairs.

Sequence data from two mt DNA segments of honey bee populations from different regions of Greece: Evros, Challkidiki, Kefalonia, Rhodes, Crete, Macedonia, Messinia,. Limnos, Nisyros, as well as from Cyprus, Italy and Albania, were used to investigate their phylogenetic relationships.

The mtDNA segments analyzed was a portion of COI and ND5, amplified using PCR after the extraction of total DNA. A total of 910bp and 688bp respectively were obtained by sequencing the above mentioned segments. CLUSTAL package was used for the alignment of these sequences. Nucleotide divergence was calculated using MEGA package and cladogram was constructed with various phylogenetic packages.

The results of our investigation show that no considerable interpopulation variability was detected.

Istituto Zooprofilattico Sperimentale delle Venezie, 35020 Legnaro (PD), Italy,

A monitoring study in a natural reserve of Northern Italy (Regional Park of Colli Euganei) was set up in 2003 and 2004 with the aim to test environment quality using honeybees as biological indicator. In particular, agricultural pollution was determined by the analysis of dead bees, whereas the presence of heavy metals in honey was used to verify air quality. The monitoring station was composed of two beehives of similar strength and equipped with “underbasket cage” to collect dead bees. Beehives were weekly monitored for strength, development, bee mortality and diseases. Dead honeybees and samples of honey were be collected, properly stored and analysed for agricultural pesticides and heavy metals (cadmium and lead) residues.

During 2003 five stations were recruited and distributed in rural areas characterised by orchards, olive-groves, vineyard and sown lands. The programme started in April and continued until September. The honeybees mortality never reached the critical threshold and the pesticides analysis resulted negative. The lead content in honey ranged from 110 and 160 ng/g, whereas the cadmium mean level was 1,5 ± 1,7 ng/g.

In 2004 nine monitoring stations were used from June to November and located either in urban or rural areas. The honeybees mortality was < 50 bee/hive/week and the pesticides analysis of dead bees resulted negative. Honey samples were taken either from super or nest. In the first case lead and cadmium mean content was 192,6 ± 118,5 ng/g and 5,2 ± 2,5 ng/g respectively, whereas in the nest honey lead residues ranged from 214 and 488 ng/g and those of cadmium from 4 e 14 ng/g. Weather conditions (poor rainfall) and stations located close to urban areas could explain the higher heavy metals content in samples collected in 2004 than 2003.

The honey bee of the eastern Mediterranean, A. mellifera syriaca, is found in the regions of Syria, Lebanon, Israel and Jordan and occupies an interesting place in the biogeography of the species as the most south-western of the oriental morphological already neighboring African bees. Its genetic integrity is endangered by persistent honey bee imports of commercial breeder lines into the region. A total of 25 samples of honey bees were collected from 14 locations throughout Jordan. Total nucleic acids of one individual worker per sample were extracted with a modified phenol-chloroform method (Arias and Sheppard 1996). A mitochondrial fragment containing the intergenic region between the tRNAleu gene and the second subunit of the cytochrome oxidase gene was amplified using the primer pair E2-H2 (Garnery et al., 1993) and digested with the restriction enzyme DraI. Restriction fragments were separated on 10% polyacrylamide gels, stained with ethidium bromide and photographed under UV illumination.

Of the 23 samples that gave positive amplifications, seven were characterized by haplotypes of the C lineage and 16 by haplotypes of the mitochondrial O lineage as described by Franck et al (2000). Haplotypes C1 and C2 were predominantly observed in the north of the country, whereas haplotypes of the O lineage were distributed throughout Jordan. One new haplotype (O3’) was found. While the presence of the C2 haplotype can be explained with natural admixture between lineages C and O in Jordan, the occurrence of the C1 haplotype strongly suggests past or recent importation of A. m.ligustica queens. Haplotypes were mostly consistent with morphometric allocation of bees to the subspecies.