Polymorphism of Locus COI-COII of Mitochondrial DNA in the Honeybee Apis mellifera L. from the Southern Ural Region

In spite of high biodiversity within the honeybee species Apis melliferaL., only one geographical race, the dark-colored forest honeybee A. m. mellifera, is uniquely adapted to severe environmental conditions of Eurasian forest and forest-steppe zones. Within the vast range of this race, only single isolates remain, where the dark forest honeybee is purebred. The Bashkir population is supposed to be one of these isolates. Molecular-genetic assessment of the state of the gene pool of this population revealed that southern honeybee races were introduced into the Bashkortostan Republic with great intensity, which was above the assimilation capacity of the population. The main part of the former range of the Bashkir population represents a hybrid zone with approximately equal ratio between gene pools of local and introduced honeybees. Our studies provide the possibility to single out one extant reserve of A. m. mellifera, Burzyanskii raion, in which the proportion of local bees in the gene pool is 0.98.     

Local honeybee (Apis mellifera mellifera L.) populations in the Urals

The COI-COII intergenic region of mitochondrial DNA (mtDNA) was studied in local honeybee (Apis mellifera mellifera) L. populations from the Middle and Southern Urals. Analysis of bee colonies in these regions revealed apiaries enriched in families descending from A. m. mellifera in the maternal lineage. These results confirm the suggestion of preservation of A. m. mellifera refuges in the Urals and provide grounds for work on the preservation of the gene pool of this bee variety, valuable for all Russia.     

Varying degrees of Apis mellifera ligustica introgression in protected populations of the black honeybee, Apis mellifera mellifera, in northwest Europe

The natural distribution of honeybee subspecies in Europe has been significantly affected by human activities during the last century. Non-native subspecies of honeybees have been introduced and propagated, so that native black honeybee (Apis mellifera mellifera) populations lost their identity by gene-flow or went extinct. After previous studies investigated the remaining gene-pools of native honeybees in France and Spain, we here assess the genetic composition of eight northwest European populations of the black honeybee, using both mitochondrial (restriction fragment length polymorphisms of the intergenic transfer RNAleu-COII region) and nuclear (11 microsatellite loci) markers. Both data sets show that A. m. mellifera populations still exist in Norway, Sweden, Denmark, England, Scotland and Ireland, but that they are threatened by gene flow from commercial honeybees. Both Bayesian admixture analysis of the microsatellite data and DraI-RFLP (restriction fragment length polymorphism) analysis of the intergenic region indicated that gene-flow had hardly occurred in some populations, whereas almost 10% introgression was observed in other populations. The most introgressed population was found on the Danish Island of Laeso, which is the last remaining native Danish population of A. m. mellifera and the only one of the eight investigated populations that is protected by law. We discuss how individual admixture analysis can be used to monitor the restoration of honeybee populations that suffer from unwanted hybridization with non-native subspecies.     

Molecular genetic analysis of five extant reserves of black honeybee <Emphasis Type="Italic">Apis melifera melifera</Emphasis> in the Urals and the Volga region

Local populations of the black honeybee Apis mellifera mellifera from the Urals and the Volga region were examined in comparison with local populations of southern honeybee subspecies A. m. caucasica and A. m. carpatica from the Caucasus and the Carpathians. Genetic analysis was performed on the basis of the polymorphism of nine microsatellite loci of nuclear DNA and the mtDNA COI–COII locus. On the territory of the Urals and the Volga region, five extant populations (reserves) of the black honeybee A. m. mellifera were identified, including the Burzyanskaya, Tatyshlinskaya, Yuzhno-Prikamskaya, Visherskaya, and Kambarskaya populations. These five populations are the basis of the modern gene pool of the black honeybee A. m. mellifera from the Urals and the Volga region. The greatest proportion of the remaining indigenous gene pool of A. m. mellifera (the core of the gene pool of the population of A. m. mellifera) is distributed over the entire territory of Perm krai and the north of the Republic of Bashkortostan. For the population of A. m. mellifera from the Urals and the Volga region, the genetic standards were calculated, which will be useful for future population studies of honeybees.     

New approach to the mitotype classification in black honeybee <Emphasis Type="Italic">Apis mellifera mellifera</Emphasis> and Iberian honeybee <Emphasis Type="Italic">Apis mellifera iberiensis</Emphasis>

The black honeybee Apis mellifera mellifera L. is today the only subspecies of honeybee which is suitable for commercial breeding in the climatic conditions of Northern Europe with long cold winters. The main problem of the black honeybee in Russia and European countries is the preservation of the indigenous gene pool purity, which is lost as a result of hybridization with subspecies, A. m. caucasica, A. m. carnica, A. m. carpatica, and A. m. armeniaca, introduced from southern regions. Genetic identification of the subspecies will reduce the extent of hybridization and provide the gene pool conservation of the black honeybee. Modern classification of the honeybee mitotypes is mainly based on the combined use of the DraI restriction endonuclease recognition site polymorphism and sequence polymorphism of the mtDNA COI–COII region. We performed a comparative analysis of the mtDNA COI–COII region sequence polymorphism in the honeybees of the evolutionary lineage M from Ural and West European populations of black honeybee A. m. mellifera and Spanish bee A. m. iberiensis. A new approach to the classification of the honeybee M mitotypes was suggested. Using this approach and on the basis of the seven most informative SNPs of the mtDNA COI–COII region, eight honeybee mitotype groups were identified. In addition, it is suggested that this approach will simplify the previously proposed complicated mitotype classification and will make it possible to assess the level of the mitotype diversity and to identify the mitotypes that are the most valuable for the honeybee breeding and rearing.     

Population structure of common juniper in the Cis-Urals and Southern Urals

The population structure of common juniper (Juniperus communis L.) growing in the Cis-Urals and Southern Urals has been studied using 17 morphological traits of generative and vegetative organs. A multivariate analysis of ten coenopopulations has recognized three phenotypically different local populations: Cis-Ural, forest Cis-Ural forest-steppe, and Southern Ural mountain populations. The Cis-Ural forest population is strictly associated with lowland pine forests of the northwestern part of the Bashkir Cis-Urals. The Cis-Ural forest-steppe population is located in the northwestern part of the Bashkir Cis-Urals and the southeastern part of the Udmurt Cis-Urals. The Southern Ural mountain population is located in the central part of the Southern Urals and is associated mainly with mountain pine and dark coniferous forests. The last population is divided into forest and forest-edge subpopulations; the first one is represented by typical undergrowth locations, whereas the second is associated with open steppelike slopes and forest edges. In general, based on morphological traits of generative organs, the revealed local subpopulations hold an intermediate position between the Eastern European and Siberian populations of common juniper. Based on the morphological traits of vegetative organs, Cis-Ural populations are considered related to the populations of the European part of Russia, whereas the mountain Southern Ural population resemble Siberian populations. Concerning morphological traits of generative organs, the intrapopulation phenotypic diversity of common juniper is higher for mountain habitats; in the case of vegetative organs, the maximum diversity is observed for lowland habitats. The character of phenotypic differentiation determines the need to conserve the gene pool of common juniper of the Cis-Urals and southern Urals on a population basis.     

Pheromonal dominance and the selection of a socially parasitic honeybee worker lineage (Apis mellifera capensis Esch.)

The recent invasion by self-replicating socially parasitic Cape honeybee workers, Apis mellifera capensis, of colonies of the neighbouring African subspecies Apis mellifera scutellata represents an opportunity to study evolution of intraspecific parasitism in real time. As honeybee workers compete pheromonally for reproductive dominance, and as A. m. capensis workers readily produce queen-like pheromones, we hypothesized that these semiochemicals promoted the evolution of intraspecific social parasitism. Remarkably, the offspring of a single worker became established as a parasite in A. m. scutellata's range. This could have resulted from extreme selection among different clonal parasitic worker lineages. Using pheromonal contest experiments, we show that the selected parasitic lineage dominates in the production of mandibular gland pheromones over all other competitors to which it is exposed. Our results suggest that mandibular gland pheromones played a key role in the evolution of intraspecific social parasitism in the honeybee and in the selection of a single genotype of parasitic workers.     

Geographical overlap of two mitochondrial genomes in Spanish honeybees (Apis mellifera iberica)

Restriction enzyme cleavage maps of mitochondrial DNA from the Spanish honeybee, Apis mellifera iberica (Hymenoptera: Apidae), were compared with those from the European subspecies A. m. mellifera, A. m. ligustica, and A. m. carnica, and the African subspecies A. m. intermissa and A. m. scutellata. The mitochondrial DNA (mtDNA) of the two African subspecies can be distinguished by restriction fragment polymorphisms revealed by Hinf I digests. Two distinct mtDNA types were found among Spanish honeybees: a west European mellifera-like type, which predominates in the north of Spain, and an African intermissa-like type, which predominates in the south. Spain appears to be a region of contact and hybridization between the two subspecies A. m. intermissa and A. m. mellifera, which respectively represent African and west European honeybee lineages. This natural boundary between European and African honeybee populations in the Old World may provide a model for predicting the eventual outcome of the colonization of North America by introduced African honeybees.     

Local honeybee (<Emphasis Type="Italic">Apis mellifera mellifera</Emphasis> L.) populations in the Urals

The COI-COII intergenic region of mitochondrial DNA (mtDNA) was studied in local honeybee (Apis mellifera mellifera) L. populations from the Middle and Southern Urals. Analysis of bee colonies in these regions revealed apiaries enriched in families descending from A. m. mellifera in the maternal lineage. These results confirm the suggestion of preservation of A. m. mellifera refuges in the Urals and provide grounds for work on the preservation of the gene pool of this bee variety, valuable for all Russia.     

Use of a PCR method for controlling pure-breeding of honeybees Apis mellifera mellifera L. in the southern Urals

A key problem of honeybee (Apis mellifera mellifera) breeding in the Southern Urals is its cross-breeding with the Caucasian honeybee Apis mellifera caucasica. Mitochondrial DNA (mtDNA) in these subspecies differ in the length of a fragment localized between genes CO-I and CO-II, which can be used as a marker. A pair of 20-mer primers for PCR was chosen by means of computer design in order to determine the fragment size in both of the subspecies. The amplified fragment was shown to have a length of 350 bp in A. m. caucasica and 600 bp in A. m. mellifera. The difference in length results from the different ratio between two main elements P and Q, which comprise a major part of this sequence in these subspecies: a copy of P element and two copies of Q element in A. m. mellifera, and a copy of Q element only in A. m. caucasica. This sharply defined distinction allows us to use PCR for differentiating the subspecies, estimating the heterogeneity in the colonies, and rejecting queens in the selection process because of the maternal inheritance of the studied character. The nucleotide sequence of the amplified mtDNA fragment of A. m. mellifera was determined.     

Fine spatial structure of allozyme genotypes in isolated population of pedunculate oak Quercus robur L. (Fagaceae)

The extent and spatial pattern of genetic variation at polymorphic allozyme loci in a population of pedunculate oak Quercus robur from the Bashkir Transural region was investigated using autocorrelation analysis. In the plantation examined, statistically significant local concentration of most of the alleles in two-dimensional space was identified. The measures for protection of this small population located outside of the western border of the species range, in the mountain--steppe habitat, and characterized by specific gene pool, are suggested.     

引入西方蜜蜂对中蜂的危害及生态影响

作者阐述自1896年中国引进西方蜜蜂Apis melliferaL.的优良品种如意大利蜂Apis mellifera ligusticaSpinola和喀尼阿兰蜂Apis mellifera Carnica Pollmann以来,使当地的东方蜜蜂Apis cerana F.受到严重危害,其分布区域缩小75%以上,种群数量减少80%以上。使山林植物授粉总量减少,导致植物多样性减少。文中提出: 建立自然保护区保存本地蜜蜂遗传特性,和采用基因转移等技术,培育具有西方蜜蜂优良生产性能的中蜂新品种,逐步恢复中蜂的种群数量。     

Racial Differences in Division of Labor in Colonies of the Honey Bee (Apis mellifera)

We measured the age at onset of foraging in colonies derived from three races of European honey bees, Apis mellifera mellifera, Apis mellifera caucasica and Apis mellifera ligustica , using a cross-fostering design that involved six unrelated colonies of each race. There was a significant effect of the race of the introduced bees on the age at onset of foraging: cohorts of A. m. ligustica bees showed the earliest onset, regardless of the race of the colony they were introduced to. There also was a significant effect of the race of the host colony: cohorts of bees introduced into mellifera colonies showed the earliest onset of foraging, regardless of the race of the bees introduced. Significant inter-trial differences also were detected, primarily because of a later onset of foraging in trials conducted during the autumn (September–October). These results demonstrate differences among European races of honey bees in one important component of colony division of labor. They also provide a starting point for analyses of the evolution of division of labor under different ecological conditions.     

Fine spatial structure of allozyme genotypes in isolated population of pedunculate oak <Emphasis Type="Italic">Quercus robur</Emphasis> L. (Fagaceae)

The extent and spatial pattern of genetic variation at polymorphic allozyme loci in a population of pedunculate oak Quercus robur from the Bashkir Transural region was investigated using autocorrelation analysis. In the plantation examined, statistically significant local concentration of most of the alleles in two-dimensional space was identified. The measures for protection of this small population located outside of the western border of the species range, in the mountain-steppe habitat, and characterized by specific gene pool, are suggested.     

Factors affecting the dynamics of the honeybee (Apis mellifera) hybrid zone of South Africa

Hybrid zones are found wherever two populations distinguishable on the basis of heritable characters overlap spatially and temporally and hybridization occurs. If hybrids have lower fitness than the parental types a tension zone may emerge, in which there is a barrier to gene flow between the two parental populations. Here we discuss a hybrid zone between two honeybee subspecies, Apis mellifera capensis and A. m. scutellata and argue that this zone is an example of a tension zone. This tension zone is particularly interesting because A. m. capensis can be a lethal social parasite of A. m. scutellata. However, despite its parasitic potential, A. m. capensis appears to be unable to increase its natural range unassisted. We propose three interlinked mechanisms that could maintain the South African honeybee hybrid zone: (1) low fitness of intercrossed and genetically mixed colonies arising from inadequate regulation of worker reproduction; (2) higher reproductive success of A. m. scutellata via both high dispersal rates into the hybrid zone and increased competitiveness of males, countered by (3) the parasitic nature of A. m. capensis.     

Hypertrehalosaemic peptides in the honeybee (Apis mellifera): purification,identification and function

The corpora cardiaca (CC) of the Italian race (including also the africanised variety) of the honeybee (Apis mellifera ligustica) contain approximately 3 pmol of a hypertrehalosaemic peptide. This peptide is identical in structure to the adipokinetic hormone (AKH) found in Manduca sexta, Mas-AKH. The CC of the dark European race of the honeybee (Apis mellifera carnica) contain no detectable Mas-AKH or any other adipokinetic/hypertrehalosaemic peptide. This is the first report of the occurrence of this peptide in a non-lepidopteran insect and of an intraspecific variation with regards to the presence or absence of a hypertrehalosaemic peptide in the CC of an insect. Extracts of A. m. ligustica CC elicit a strong adipokinetic/hypertrehalosaemic response when injected into crickets and cockroaches but extracts of A. m. carnica CC elicit no such responses when injected into crickets, cockroaches and butterflies. A weak hypertrehalosaemic response to injected Mas-AKH was observed in winter bees of both races, but there was no response in spring/summer bees. However, if a seasonal difference exists, it is at best minimal. Honeybees always have access to a more than adequate supply of high energy food in the form of nectar or honey stored in the hive. Thus, though A. m. ligustica CC contain a hypertrehalosaemic peptide, there is neither a glycogen-mobilising function of this hormone nor an adequate glycogen store in their fat body for its effective utilisation.     

Further evidence of an oriental origin for Nosema ceranae (Microsporidia: Nosematidae)

Although Nosema ceranae was first isolated from the Asian honeybee (Apis cerana) in Asia and then subsequently recognized as a widespread gut parasite of the Western honeybee (Apis mellifera), its origins and primary host are yet to be accurately established. In this study we examined the possibility of an Asian origin for the parasite by looking for evidence of its ongoing spread out of Asia. To do this, we surveyed for the presence of N. ceranae in A. cerana and A. mellifera on isolated islands of the Solomon Islands (Pacific region), most of which were inhabited with A. mellifera that had been introduced from Australia and New Zealand at a time when N. ceranae was not present in either country, but on which some had also recently become inhabited with invasive A. cerana that originated from Asia with no prior history of contact with A. mellifera infected with N. ceranae. We also sought to verify previous findings that N. ceranae was widespread in Asian honeybees by surveying for its presence in isolated populations of the Asian honeybees, A. cerana, A. koschevnikovi, A. nigrocincta and A. florea. We obtained evidence that A. cerana introduced N. ceranae to A. mellifera in the Solomon Islands and also confirmed the widespread occurrence of the parasite in Asian honeybees, even reporting it for the first time in A. koschevnikovi from Borneo. Our findings provide further support for the hypothesis that N. ceranae has only recently emerged from Asia to become a parasite of A. mellifera.     

Allozyme Variability and Phylogenetic Relationships in Honey Bee (Hymenoptera: Apidae: Apis mellifera) Populations From Greece and Cyprus

Ten gene enzymic systems (alpha-GPDH, AO, MDH, ADH, LAP, SOD, ALP, ACPH, ME, and EST), corresponding to 12 genetic loci, were assayed from five Greek populations representing three subspecies of Apis mellifera, A. m. cecropia (Pthiotida, Kythira), A. m. macedonica (Macedonia), and the "Aegean race" of A. mellifera, which is supposed to be very similar to A. m. adami (Ikaria, Kasos), as well as a population from Cypus (A. m. cypria). ADH( *)-1, ADH( *)-2, and LAP( *) electrophoretic patterns discriminate the Cyprus population from the Greek populations. MDH( *)-1, EST( *)-3, SOD( *), ALP( *), and ME( *) loci were found to be polymorphic in almost all populations. The observed heterozygosity was found to range from 0.066 to 0.251. Allele frequencies of all loci were used to estimate Nei's genetic distance, which was found to range between 0.011 and 0.413 among the populations studied. UPGMA and neighbor-joining phylogenetic trees obtained by genetic distance matrix methods, as well as a Wagner tree based on the discrete character parsimony method, support the hypothesis that the most distant population is that from Cyprus. Our allozymic data support A. m. cypria as a distinct subspecies, but there was no allozymic support for the distinction of the other subspecies existing in Greece.     

Widespread dispersal of the microsporidian Nosema ceranae, an emergent pathogen of the western honey bee, Apis mellifera

The economically most important honey bee species, Apis mellifera, was formerly considered to be parasitized by one microsporidian, Nosema apis. Recently, [Higes, M., Martín, R., Meana, A., 2006. Nosema ceranae, a new microsporidian parasite in honeybees in Europe, J. Invertebr. Pathol. 92, 93-95] and [Huang, W.-F., Jiang, J.-H., Chen, Y.-W., Wang, C.-H., 2007. A Nosema ceranae isolate from the honeybee Apis mellifera. Apidologie 38, 30-37] used 16S (SSU) rRNA gene sequences to demonstrate the presence of Nosema ceranae in A. mellifera from Spain and Taiwan, respectively. We developed a rapid method to differentiate between N. apis and N. ceranae based on PCR-RFLPs of partial SSU rRNA. The reliability of the method was confirmed by sequencing 29 isolates from across the world (N =9 isolates gave N. apis RFLPs and sequences, N =20 isolates gave N. ceranae RFLPs and sequences; 100% correct classification). We then employed the method to analyze N =115 isolates from across the world. Our data, combined with N =36 additional published sequences demonstrate that (i) N. ceranae most likely jumped host to A. mellifera, probably within the last decade, (ii) that host colonies and individuals may be co-infected by both microsporidia species, and that (iii) N. ceranae is now a parasite of A. mellifera across most of the world. The rapid, long-distance dispersal of N. ceranae is likely due to transport of infected honey bees by commercial or hobbyist beekeepers. We discuss the implications of this emergent pathogen for worldwide beekeeping.     

Cautions on direct gene flow estimation in plant populations

Through simulations we have investigated the statistical properties of two of the main approaches for directly estimating pollen gene flow (m) in plant populations: genotypic exclusion and mating models. When the assumptions about accurately known background pollen pool allelic frequencies are met, both methods provide unbiased results with comparable variances across a range of true m values. However, when presumed allelic frequencies differ from actual ones, which is more likely in research practice, both estimators are biased. We demonstrate that the extent and direction of bias largely depend on the difference (measured as genetic distance) between the presumed and actual pollen pools, and on the degree of genetic differentiation between the local population and the actual background pollen sources. However, one feature of the mating model is its ability to estimate pollen gene flow simultaneously with background pollen pool allelic frequencies. We have found that this approach gives nearly unbiased pollen gene flow estimates, and is practical because it eliminates the necessity of providing independent estimates of background pollen pool allelic frequencies. Violations of the mating model assumptions of random mating within local population affect the precision of the estimates only to a limited degree.