SummaryThe natural diversity of honey bees in Europe is eroding fast. A multitude of reasons lead to a loss of both genetic diversity and specific adaptations to local conditions. To preserve locally adapted bees through breeding efforts and to maintain regional strains in conservation areas, these valuable populations need to be identified.In this paper, we give an overview of methods that are currently available and used for recognition of honey bee subspecies and ecotypes, or that can be utilised to verify the genetic origin of colonies for breeding purposes. Beyond summarising details of morphometric, allozyme and DNA methods currently in use, we report recommendations with regard to strategies for sampling, and suggest methods for statistical data analysis. In particular, we emphasise the importance of reference data and consistency of methods between laboratories to yield comparable results. Métodos estándar para la caracterización de las subespecies y ecotipos de Apis mellifera ResumenLa diversidad natural de la abeja de la miel se está deteriorando rápidamente en Europa. Existen multitud de razones que conducen tanto a una pérdida de diversidad genética como de adaptaciones específicas a las condiciones locales. Se necesita identificar a estas valiosas poblaciones para preservar a las abejas adaptadas a nivel local, mediante esfuerzos para la mejora y el mantenimiento de variedades regionales en las áreas de conservación.En este artículo, realizamos una revisión general de los actuales métodos disponibles que se utilizan para la determinación de subespecies y ecotipos de abejas melíferas, o que pueden ser utilizados para verificar el origen genético de las colmenas seleccionadas con fines de cría.Además, resumimos las características de los métodos morfométricos, de aloenzimas y de ADN, realizamos recomendaciones con respecto a las estrategias de muestreo, y sugerimos métodos para el análisis estadístico de los datos. En particular, destacamos la importancia de los datos de referencia y la coherencia de los métodos entre laboratorios para producir resultados comparables.

SummaryFrom 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: Métodos estándar para la investigación molecular en Apis mellifera ResumenLas abejas de miel han sido durante mucho tiempo un organismo clave para avances fundamentales en biología a partir de estudios de su comportamiento, comunicación química, genómica y de biología del desarrollo, entre otros muchos. Con la secuencia del genoma en la mano y herramientas genéticas mucho mejores, las abejas son ahora un blanco aún más atractivo para responder a las preguntas más importantes de la biología evolutiva, la estructura de las poblaciones y la organización social. Al mismo tiempo, los incentivos agrícolas para entender cómo las abejas caen enfermas, o evadir y sobrevivir a sus muchas plagas y patógenos, han presionado para comprender genéticamente la inmunidad individual y social en esta especie. A continuación se describen y se hace referencia a herramientas que hacen uso de modernas técnicas de biología molecular para entender el comportamiento de las abejas, su salud y otros aspectos de su biología. Nos centramos en las técnicas de ADN y ARN, en gran parte debido a que las técnicas de evaluación de las proteínas de la abeja se tratan en detalle en Hartfelder et al. (2013). Cubrimos las necesidades prácticas de toma de muestras de abejas, su transporte y almacenamiento, y luego se discuten una serie de técnicas actuales de análisis genético. A continuación, se proporciona una hoja de ruta para los recursos genómicos y métodos para estudiar las abejas, seguido de protocolos estadísticos específicos de la genética de poblaciones, la genética cuantitativa y la filogenia.Finalmente, se termina con tres herramientas importantes para predecir la regulación génica y la fu...

SummaryThe 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 tRNA leu -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. 270Pinto et al.

Nosema ceranae is a hot topic in honey bee health as reflected by numerous papers published every year. This review presents an update of the knowledge generated in the last 12 years in the field of N. ceranae research, addressing the routes of transmission, population structure and genetic diversity. This includes description of how the infection modifies the honey bee's metabolism, the immune response and other vital functions. The effects on individual honey bees will have a direct impact on the colony by leading to losses in the adult's population. The absence of clear clinical signs could keep the infection unnoticed by the beekeeper for long periods. The influence of the environmental conditions, beekeeping practices, bee genetics and the interaction with pesticides and other pathogens will have a direct influence on the prognosis of the disease. This review is approached from the point of view of the Mediterranean countries where the professional beekeeping has a high representation and where this pathogen is reported as an important threat.

The natural distribution of the honeybee (Apis mellifera L.) has been changed by humans in recent decades to such an extent that the formerly widest-spread European subspecies, Apis mellifera mellifera, is threatened by extinction through introgression from highly divergent commercial strains in large tracts of its range. Conservation efforts for A. m. mellifera are underway in multiple European countries requiring reliable and cost-efficient molecular tools to identify purebred colonies. Here, we developed four ancestry-informative SNP assays for high sample throughput genotyping using the iPLEX Mass Array system. Our customized assays were tested on DNA from individual and pooled, haploid and diploid honeybee samples extracted from different tissues using a diverse range of protocols. The assays had a high genotyping success rate and yielded accurate genotypes. Performance assessed against whole-genome data showed that individual assays behaved well, although the most accurate introgression estimates were obtained for the four assays combined (117 SNPs). The best compromise between accuracy and genotyping costs was achieved when combining two assays (62 SNPs). We provide a ready-to-use cost-effective tool for accurate molecular identification and estimation of introgression levels to more effectively monitor and manage A. m. mellifera conservatories.

Beekeeping activities, especially queen trading, have shaped the distribution of honey bee (Apis mellifera) subspecies in Europe, and have resulted in extensive introductions of two eastern European C-lineage subspecies (A. m. ligustica and A. m. carnica) into the native range of the M-lineage A. m. mellifera subspecies in Western Europe. As a consequence, replacement and gene flow between native and commercial populations have occurred at varying levels across western European populations. Genetic identification and introgression analysis using molecular markers is an important tool for management and conservation of honey bee subspecies. Previous studies have monitored introgression by using microsatellite, PCR-RFLP markers and most recently, high density assays using single nucleotide polymorphism (SNP) markers. While the latter are almost prohibitively expensive, the information gained to date can be exploited to create a reduced panel containing the most ancestry-informative markers (AIMs) for those purposes with very little loss of information. The objective of this study was to design reduced panels of AIMs to verify the origin of A. m. mellifera individuals and to provide accurate estimates of the level of C-lineage introgression into their genome. The discriminant power of the SNPs using a variety of metrics and approaches including the Weir & Cockerham’s FST, an FST-based outlier test, Delta, informativeness (In), and PCA was evaluated. This study shows that reduced AIMs panels assign individuals to the correct origin and calculates the admixture level with a high degree of accuracy. These panels provide an essential tool in Europe for genetic stock identification and estimation of admixture levels which can assist management strategies and monitor honey bee conservation programs.

A large-scale survey of the Iberian honey bee (Apis mellifera iberiensis) diversity patterns, using sequence data of the tRNA leu-cox2 mitochondrial DNA (mtDNA) region, demonstrates that earlier studies based on the Dra I test missed significant components of genetic variation. Based on results from this survey, existing haplotype names were revised and updated following a nomenclature system established earlier and extended herein for the intergenic region. A more complete picture of the complex diversity patterns of IHBs is revealed that includes 164 novel haplotypes, 113 belonging to lineage A and 51 to lineage M and within lineage A and 69 novel haplotypes that belong to sub-lineage A I , 13 to A II , and 31 to A III. Within lineage M, two novel haplotypes show a striking architecture with features of lineages A and M, which based on sequence comparisons and relationships among haplotypes are seemingly ancestral. These data expand our knowledge of the complex architecture of the tRNA leu-cox2 intergenic region in Apis mellifera and re-emphasizes the importance of Iberia as a source of honey bee mtDNA diversity. Iberian honey bee / Apis mellifera intermissa / ancestral haplotype M / Dra I test

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.