Pilar De la Rúa

Biodiversity, conservation and current threats to European honeybees

Europe harbours several endemic honeybee (Apis mellifera) subspecies. Yet the distribution of these subspecies is nowadays also much influenced by beekeeping activities. Large scale migratory beekeeping and trade in queens, coupled with the promiscuous mating system of honeybees, have exposed native European honeybees to increasing introgressive hybridization with managed non-native subspecies, which may lead to the loss of valuable combinations of traits shaped by natural selection. Other threats to European honeybees are factors that have caused a progressive decline in A. mellifera throughout the world in recent years, leading to large economic losses and jeopardizing ecosystem functioning. We review the biodiversity of European honeybees and summarize the management and conservation strategies employed by different countries. A comprehensive picture of the beekeeping industry in Europe is also provided. Finally we evaluate the potential threats affecting the biodiversity of European honeybee populations and provide some perspectives for future research.ZusammenfassungDie Unterarten der Honigbienen wurden sowohl mit morphologischen (Box 1) als auch mit molekulargenetischen (Box 2 und 3) Methoden untersucht. Die in Europa vorkommenden elf Unterarten (Abb. 1) werden in vier evolutiven Abstammungslinien eingeteilt. In den entsprechenden Verbreitungsgebieten sind die dazugehörenden Unterarten unterschiedlichen Klima- und Habitatsbedingungen sowie anthropogenen Einflüssen ausgesetzt. Unser erstes Ziel ist es, die Biodiversität der europäischen Honigbienen zu beschreiben und die Strategien zum Schutz der Honigbienen in den einzelnen Ländern zusammenzufassen. Hybridisierungsprozesse wurden vor allem auf der iberischen, italienischen und der Balkan-Halbinsel festgestellt, wohingegen natürliche (aufgrund von Genfluss durch die Mehrfachpaarung der Königin) und durch imkerliche Aktivitäten ausgelöste (durch die Einfuhr von Honigbienen-Unterarten außerhalb ihres natürlichen Verbreitungsgebietes) genetische Introgression in Zentral- und Osteuropa sowie auf Mittelmeerinseln beobachtet wurden. Verschiedene Naturschutzprogramme wurden auf europäischen Inseln (Dänemark, Spanien) und seit kurzem auch in anderen europäischen Ländern (Frankreich, Norwegen, Slowenien und Österreich) etabliert. Für einen sinnvollen Honigbienenschutz muss aber der Status der imkerlich gehaltenen Honigbienenpopulation in den jeweiligen Ländern mit berücksichtigt werden. Daher müssen zunächst detaillierte Informationen zur Imkerei in den einzelnen Ländern gesammelt werden, bevor zukünftige Naturschutzprogramme entwickelt werden (Abb. 2 und Tab. I in „supplementary data“). Auf dieser Grundlage werden zwei Hauptansätze für zukünftige Naturschutzrichtlinien vorgeschlagen: Beschränkung der Einfuhr von „überlegenen“ Unterarten in Gebiete, die bereits von nativen Honigbienenpopulationen besetzt sind sowie die Aufrechterhaltung der genetischen Diversität in natürlichen Honigbienenpopulationen. Immer mehr Faktoren wie veränderte Landnutzung, die Verbreitung von Krankheitserregern und Parasiten, der Einsatz von Pestiziden und Herbiziden (Tab. I) bedrohen die Honigbienen in Europa und gefährden damit auch die Funktion des Ökosystems durch eine unzureichende Bestäubung von Wild- und Kulturpflanzen.Das vor kurzem aufgeschlüsselte Honigbienen-Genom bietet nun aber neue Möglichkeiten, auf molekularer Ebene die Genetik, Physiologie und das Verhalten der Honigbienen zu untersuchen. Molekulare Marker wie SNPs („Single Nucleotide Polymorphisms“) und Mikrosatelliten ermöglichen neue Einblicke in die Populationsstruktur der Honigbienen und die Analyse des Honigbienen-Proteoms wird uns zusätzlich Informationen über die Struktur, Funktion und Wechselwirkungen der von den jeweiligen Genen produzierten Proteine geben.Eine abschließende Überlegung ist, dass der Honigbienenschutz eng mit der Aufrechterhaltung der Imkerei verbunden ist, die als zukunftsträchtiger Bestandteil der landwirtschaftlichen Praxis auch für die junge Generation attraktiv sein sollte. Für eine nachhaltige Unterstützung der Imkerei sollten die Berufsausbildung verbessert, moderne Betriebsweisen eingeführt, angewandte Forschung zur Bienenbiologie, Genetik und Krankheitsbekämpfung durchgeführt sowie sinnvolle Richtlinien zum Schutz wertvoller Ökosysteme umgesetzt werden.

A worldwide survey of genome sequence variation provides insight into the evolutionary history of the honeybee Apis mellifera.

The honeybee Apis mellifera has major ecological and economic importance. We analyze patterns of genetic variation at 8.3 million SNPs, identified by sequencing 140 honeybee genomes from a worldwide sample of 14 populations at a combined total depth of 634×. These data provide insight into the evolutionary history and genetic basis of local adaptation in this species. We find evidence that population sizes have fluctuated greatly, mirroring historical fluctuations in climate, although contemporary populations have high genetic diversity, indicating the absence of domestication bottlenecks. Levels of genetic variation are strongly shaped by natural selection and are highly correlated with patterns of gene expression and DNA methylation. We identify genomic signatures of local adaptation, which are enriched in genes expressed in workers and in immune system– and sperm motility–related genes that might underlie geographic variation in reproduction, dispersal and disease resistance. This study provides a framework for future investigations into responses to pathogens and climate change in honeybees.

Standard methods for molecular research in Apis mellifera

Summary From studies of behaviour, chemical communication, genomics and developmental biology, among many others, honey bees have long been a key organism for fundamental breakthroughs in biology. With a genome sequence in hand, and much improved genetic tools, honey bees are now an even more appealing target for answering the major questions of evolutionary biology, population structure, and social organization. At the same time, agricultural incentives to understand how honey bees fall prey to disease, or evade and survive their many pests and pathogens, have pushed for a genetic understanding of individual and social immunity in this species. Below we describe and reference tools for using modern molecular-biology techniques to understand bee behaviour, health, and other aspects of their biology. We focus on DNA and RNA techniques, largely because techniques for assessing bee proteins are covered in detail in Hartfelder et al. (2013). We cover practical needs for bee sampling, transport, and storage, and then discuss a range of current techniques for genetic analysis. We then provide a roadmap for genomic resources and methods for studying bees, followed by specific statistical protocols for population genetics, quantitative genetics, and phylogenetics. Finally, we end with three important tools for predicting gene regulation and function in honey bees: Fluorescence in situ hybridization (FISH), RNA interference (RNAi), and the estimation of chromosomal methylation and its role in epigenetic gene regulation.

A review of methods for discrimination of honey bee populations as applied to European beekeeping

Summary Here, scientists from 19 European countries, most of them collaborating in Working Group 4: “Diversity and Vitality” of COST Action FA 0803 “Prevention of honey bee COlony LOSSes” (COLOSS), review the methodology applied in each country for discriminating between honey bee populations. Morphometric analyses (classical and geometric) and different molecular markers have been applied. Even if the approach has been similar, however, different methodologies regarding measurements, landmarks or molecular markers may have been used, as well as different statistical procedures. There is therefore the necessity to establish common methods in all countries in order to have results that can be directly compared. This is one of the goals of WG4 of the COLOSS project.

Genetic structure of Balearic honeybee populations based on microsatellite polymorphism

The genetic variation of honeybee colonies collected in 22 localities on the Balearic Islands (Spain) was analysed using eight polymorphic microsatellite loci. Previous studies have demonstrated that these colonies belong either to the African or west European evolutionary lineages. These populations display low variability estimated from both the number of alleles and heterozygosity values, as expected for the honeybee island populations. Although genetic differentiation within the islands is low, significant heterozygote deficiency is present, indicating a subpopulation genetic structure. According to the genetic differentiation test, the honeybee populations of the Balearic Islands cluster into two groups: Gimnesias (Mallorca and Menorca) and Pitiusas (Ibiza and Formentera), which agrees with the biogeography postulated for this archipelago. The phylogenetic analysis suggests an Iberian origin of the Balearic honeybees, thus confirming the postulated evolutionary scenario for Apis mellifera in the Mediterranean basin. The microsatellite data from Formentera, Ibiza and Menorca show that ancestral populations are threatened by queen importations, indicating that adequate conservation measures should be developed for protecting Balearic bees.

Genetic integrity of the Dark European honey bee (Apis mellifera mellifera) from protected populations: a genome-wide assessment using SNPs and mtDNA sequence data

Summary The recognition that the Dark European honey bee, Apis mellifera mellifera, is increasingly threatened in its native range has led to the establishment of conservation programmes and protected areas throughout western Europe. Previous molecular surveys showed that, despite management strategies to preserve the genetic integrity of A. m. mellifera, protected populations had a measurable component of their gene pool derived from commercial C-lineage honey bees. Here we used both sequence data from the tRNAleu-cox2 intergenic mtDNA region and a genome-wide scan, with over 1183 single nucleotide polymorphisms (SNPs), to assess genetic diversity and introgression levels in several protected populations of A. m. mellifera, which were then compared with samples collected from unprotected populations. MtDNA analysis of the protected populations revealed a single colony bearing a foreign haplotype, whereas SNPs showed varying levels of introgression ranging from virtually zero in Norway to about 14% in Denmark. Introgression overall was higher in unprotected (30%) than in protected populations (8%), and is reflected in larger SNP diversity levels of the former, although opposite diversity levels were observed for mtDNA. These results suggest that, despite controlled breeding, some protected populations still require adjustments to the management strategies to further purge foreign alleles, which can be identified by SNPs.

Population genetic structure of coastal Croatian honeybees (Apis mellifera carnica)

The genetic structure and molecular diversity of Croatian honeybee coastal populations have been investigated with microsatellite and mitochondrial markers. According to sequence data of the mitochondrial tRNAleu-cox2 intergenic region, all analysed samples belong to the Central Mediterranean and Southeast European evolutionary C-lineage. Four mitochondrial haplotypes have been found in the Croatian honeybees, whereas two newly described have been found in Croatia and Greek respectively. Through the Bayesian analysis of microsatellite variation, two groups can be distinguished within the Croatian honeybee population, suggesting the existence of two subpopulations of A. m. carnica. The relation of these subpopulations with previously described ecotypes and regional variations is discussed. These results emphasize the importance of sequencing in the description of new haplotypes and therefore, in the inference of molecular biodiversity within honeybee populations. The description of two subpopulations in coastal Croatian honeybees must be considered in future conservation strategies.ZusammenfassungDas Ziel dieser Untersuchung ist eine Einschätzung der genetischen Variabilität der kroatischen Honigbiene und die Suche nach molekularen Belegen für bereits auf der Basis von morphometrischen und ökologischen Daten beschriebene Ökotypen und regionale Variationen (Ruttner, 1992). Zu Vergleichszwecken wurden Proben aus Italien und Griechenland ebenfalls analysiert.Zur Erforschung der Biogeographie von Apis mellifera wird im wesentlichen die Analyse der Variabilität der mitochondrialen DNA herangezogen, während die Struktur von Populationen aus der Analyse von Mikrosatelliten abgeleitet wird. Die anerkannten 29 Unterarten der Honigbiene (Engel, 1999; Sheppard und Meixner, 2003) wurden in fünf evolutionäre Linien eingruppiert (Garnery et al., 1992; Estoup et al., 1995; Franck et al., 2000b; Whitfield et al., 2006), von denen vier natürlicherweise im Mittelmeerbecken vorkommen: M (West- und Nordeuropa), C (Zentrales Mittelmeer und Südosteuropa), O (Naher Osten) und A (Afrika).Fünfundvierzig Bienenvölker aus Kroatien, Italien und Griechenland wurden untersucht (Tab. I und Abb. 1), die nach Ergebnissen von Sequenzdaten der mitochondrialen tRNAleu-cox2 Region alle zur mitochondrialen C-Linie gehören. Zwei neue mtDNA Haplotypen, C2e und C2i wurden in Kroatien, bzw. in Griechenland, gefunden, während alle italienischen Proben den C1 Haplotypen aufwiesen. In Kroatien wurden vier Haplotypen mit unterschiedlichen Häufigkeiten nachgewiesen: C1 (0.35), C2c (0.15), C2d (0.05), und C2e (0.45). In Griechenland war der Haplotyp C2d (0.80) häufiger als Typ C2i (0.20) (Abb. 1).In der Bayesschen Analyse der Mikrosatelliten auf der Basis von vier Gruppen wurde die kroatische Bienenpopulation in zwei Untergruppen mit einer mehr nördlichen bzw. mehr südlichen Verbreitung aufgeteilt (Kroatien-1 = 51,7 % und Kroatien-2 = 39,9 %), wobei jedoch 6,3 % der kroatischen Bienen der italienischen Population zugeordnet wurden (Tab. III und Abb. 3). Die PCA Analysen zeigten, dass sich bei Berücksichtigung von vier Gruppen die kroatischen Subpopulationen unterschiedlich zuordnen, wobei Subpopulation-2 eher den italienischen Bienen und Subpopulation-1 eher einigen griechischen Proben angenähert war (Abb. 4). Genetische Introgression aus der benachbarten A. m. ligustica in Italien wurde beobachtet, da 14,7 % der kroatischen Subpopulation-2 der italienischen Population zugeordnet wurden (Abb. 3).Der C2c Haplotyp wurde auch in Slowenien gefunden (Sušnik et al., 2004), womit eine enge Verwandtschaft zwischen der kroatischen und slowenischen Population teilweise bestätigt wurde. Dies geht wahrscheinlich auf den beidseitigen Austausch von Bienenmaterial zwischen Imkern zurück. Aus dem Auftreten von zwei Haplotypen, die für andere Unterarten charakteristisch sind, werden Einkreuzungseffekte abgeleitet, die entweder auf natürliche Weise zustande kommen, oder auf menschlichen Einfluss durch Königinnenhandel zurückgehen. Die Anwesenheit von zwei verschiedenen kroatischen Haplotypen in der dalmatischen Region wurde gezeigt: Subpopulation-1 kommt in nördlicheren Gebieten vor, während Subpopulation-2 im südlichen Teil des Gebiets auftritt. Die molekulare Analyse der kroatischen Honigbienen belegt die Notwendigkeit für ähnliche molekulare Untersuchungen von Bienenpopulationen in benachbarten Regionen, wo noch größere Vorkommen der autochthonen Carnica-Biene existieren.

The growing prevalence of Nosema ceranae in honey bees in Spain, an emerging problem for the last decade

Microsporidiosis caused by infection with Nosema apis or Nosema ceranae has become one of the most widespread diseases of honey bees and can cause important economic losses for beekeepers. Honey can be contaminated by spores of both species and it has been reported as a suitable matrix to study the field prevalence of other honey bee sporulated pathogens. Historical honey sample collections from the CAR laboratory (Centro Apícola Regional) were analyzed by PCR to identify the earliest instance of emergence, and to determine whether the presence of Nosema spp. in honey was linked to the spread of these microsporidia in honey bee apiaries. A total of 240 frozen honey samples were analyzed by PCR and the results compared with rates of Nosema spp. infection in worker bee samples from different years and geographical areas. The presence of Nosema spp. in hive-stored honey from naturally infected honey bee colonies (from an experimental apiary) was also monitored, and although collected honey bees resulted in a more suitable sample to study the presence of microsporidian parasites in the colonies, a high probability of finding Nosema spp. in their hive-stored honey was observed. The first honey sample in which N. ceranae was detected dates back to the year 2000. In subsequent years, the number of samples containing N. ceranae tended to increase, as did the detection of Nosema spp. in adult worker bees. The presence of N. ceranae as early as 2000, long before generalized bee depopulation and colony losses in 2004 may be consistent with a long incubation period for nosemosis type C or related with other unknown factors. The current prevalence of nosemosis, primarily due to N. ceranae, has reached epidemic levels in Spain as confirmed by the analysis of worker honey bees and commercial honey.

Signatures of selection in the Iberian honey bee (Apis mellifera iberiensis) revealed by a genome scan analysis of single nucleotide polymorphisms.

Understanding the genetic mechanisms of adaptive population divergence is one of the most fundamental endeavours in evolutionary biology and is becoming increasingly important as it will allow predictions about how organisms will respond to global environmental crisis. This is particularly important for the honey bee, a species of unquestionable ecological and economical importance that has been exposed to increasing human‐mediated selection pressures. Here, we conducted a single nucleotide polymorphism (SNP)‐based genome scan in honey bees collected across an environmental gradient in Iberia and used four FST‐based outlier tests to identify genomic regions exhibiting signatures of selection. Additionally, we analysed associations between genetic and environmental data for the identification of factors that might be correlated or act as selective pressures. With these approaches, 4.4% (17 of 383) of outlier loci were cross‐validated by four FST‐based methods, and 8.9% (34 of 383) were cross‐validated by at least three methods. Of the 34 outliers, 15 were found to be strongly associated with one or more environmental variables. Further support for selection, provided by functional genomic information, was particularly compelling for SNP outliers mapped to different genes putatively involved in the same function such as vision, xenobiotic detoxification and innate immune response. This study enabled a more rigorous consideration of selection as the underlying cause of diversity patterns in Iberian honey bees, representing an important first step towards the identification of polymorphisms implicated in local adaptation and possibly in response to recent human‐mediated environmental changes.

Biodiversity of Apis mellifera populations from Tenerife (Canary Islands) and hybridisation with East European races

The biodiversity of honeybee (Apis mellifera) populations from Tenerife (Canary Islands, Spain) has been assessed by restriction analysis of a mitochondrial non-coding intergenic region. Seventy-nine colonies were analysed from thirteen apiaries in six populations that have been kept from recent queen introduction. The length and restriction pattern of the PCR amplified products of the intergenic region identified four mitochondrial haplotypes. One of these haplotypes shows the same restriction pattern and composition of the intergenic region carried by honeybees belonging to the African lineage. Two haplotypes are characterised by a particular intergenic region found with high frequency in the Canarian populations. The haplotype representative of the East European honeybee lineage shows a frequency of 35%, thus indicating introduction of queen honeybees. The finding of this haplotype in Canarian honeybees suggests that hybridisation between the endemic Apis mellifera populations and imported bees is occurring in Tenerife.