Emerging diseases are among the greatest threats to honey bees. Unfortunately, where and when an emerging disease will appear are almost impossible to predict. The arrival of the parasitic Varroa mite into the Hawaiian honey bee population allowed us to investigate changes in the prevalence, load, and strain diversity of honey bee viruses. The mite increased the prevalence of a single viral species, deformed wing virus (DWV), from ~10 to 100% within honey bee populations, which was accompanied by a millionfold increase in viral titer and a massive reduction in DWV diversity, leading to the predominance of a single DWV strain. Therefore, the global spread of Varroa has selected DWV variants that have emerged to allow it to become one of the most widely distributed and contagious insect viruses on the planet.

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SummaryHoney bee virus research is an enormously broad area, ranging from subcellular molecular biology through physiology and behaviour, to individual and colony-level symptoms, transmission and epidemiology. The research methods used in virology are therefore equally diverse.This article covers those methods that are very particular to virological research in bees, with numerous cross-referrals to other BEEBOOK papers on more general methods, used in virology as well as other research. At the root of these methods is the realization that viruses at their most primary level inhabit a molecular, subcellular world, which they manipulate and interact with, to produce all higher order phenomena associated with virus infection and disease. Secondly, that viruses operate in an exponential world, while the host operates in a linear world and that much of the understanding and management of viruses hinges on reconciling these fundamental mathematical differences between virus and host. The article concentrates heavily on virus propagation and methods for detection, with minor excursions into surveying, sampling management and background information on the many viruses found in bees. Métodos estándar para la investigación de virus en Apis mellifera ResumenLa investigación de los virus de la abeja de la miel es un área sumamente amplia, que abarca desde la biología molecular subcelular hasta la fisiología y el comportamiento, desde síntomas al nivel de individuo hasta al nivel de la colmena, transmisión y epidemiología. Los métodos de investigación en virología son, por tanto, diversos. Este artículo incluye aquellos métodos específicos de la investigación virológica en las abejas, con numerosas referencias cruzadas con otros artículos del BEEBOOK y otros más generales, usados tanto en virología como en otras disciplinas. La base de estos métodos es la comprensión de los virus en su nivel primario de hábitat molecular, ambiente subcelular, que manipulan y con el que interactúan, para producir otros fenómenos de orden superior asociados a la infección del virus y la enfermedad. En segundo lugar, estos virus actúan en un mundo exponencial, mientras que los hospedadores actúan en un mundo lineal y gran parte del entendimiento y manejo de los virus depende de los fundamentos matemáticos de las diferencias entre el virus y el hospedador. El artículo se centra principalmente en la propagación de virus y en los métodos para su detección, con inclusiones menores en su estudio, el manejo del muestreo y la información general sobre los numerosos virus que se encuentran en las abejas.

Many pollinator populations are declining, with large economic and ecological implications. Parasites are known to be an important factor in the some of the population declines of honey bees and bumblebees, but little is known about the parasites afflicting most other pollinators, or the extent of interspecific transmission or vectoring of parasites. Here we carry out a preliminary screening of pollinators (honey bees, five species of bumblebee, three species of wasp, four species of hoverfly and three genera of other bees) in the UK for parasites. We used molecular methods to screen for six honey bee viruses, Ascosphaera fungi, Microsporidia, and Wolbachia intracellular bacteria. We aimed simply to detect the presence of the parasites, encompassing vectoring as well as actual infections. Many pollinators of all types were positive for Ascosphaera fungi, while Microsporidia were rarer, being most frequently found in bumblebees. We also detected that most pollinators were positive for Wolbachia, most probably indicating infection with this intracellular symbiont, and raising the possibility that it may be an important factor in influencing host sex ratios or fitness in a diversity of pollinators. Importantly, we found that about a third of bumblebees (Bombus pascuorum and Bombus terrestris) and a third of wasps (Vespula vulgaris), as well as all honey bees, were positive for deformed wing virus, but that this virus was not present in other pollinators. Deformed wing virus therefore does not appear to be a general parasite of pollinators, but does interact significantly with at least three species of bumblebee and wasp. Further work is needed to establish the identity of some of the parasites, their spatiotemporal variation, and whether they are infecting the various pollinator species or being vectored. However, these results provide a first insight into the diversity, and potential exchange, of parasites in pollinator communities.

Reports of honey bee population decline has spurred many national efforts to understand the extent of the problem and to identify causative or associated factors. However, our collective understanding of the factors has been hampered by a lack of joined up trans-national effort. Moreover, the impacts of beekeeper knowledge and beekeeping management practices have often been overlooked, despite honey bees being a managed pollinator. Here, we established a standardised active monitoring network for 5 798 apiaries over two consecutive years to quantify honey bee colony mortality across 17 European countries. Our data demonstrate that overwinter losses ranged between 2% and 32%, and that high summer losses were likely to follow high winter losses. Multivariate Poisson regression models revealed that hobbyist beekeepers with small apiaries and little experience in beekeeping had double the winter mortality rate when compared to professional beekeepers. Furthermore, honey bees kept by professional beekeepers never showed signs of disease, unlike apiaries from hobbyist beekeepers that had symptoms of bacterial infection and heavy Varroa infestation. Our data highlight beekeeper background and apicultural practices as major drivers of honey bee colony losses. The benefits of conducting trans-national monitoring schemes and improving beekeeper training are discussed.

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...

SummaryAmerican foulbrood is one of the most devastating diseases of the honey bee. It is caused by the spore-forming, Gram-positive rod-shaped bacterium Paenibacillus larvae. The recent updated genome assembly and annotation for this pathogen now permits in-depth molecular studies. In this paper, selected techniques and protocols for American foulbrood research are provided, mostly in a recipe-like format that permits easy implementation in the laboratory. Topics covered include: working with Paenibacillus larvae, basic microbiological techniques, experimental infection, and "'omics" and other sophisticated techniques. Further, this chapter covers other technical information including biosafety measures to guarantee the safe handling of this pathogen. Métodos para la investigación de la loque americana ResumenLa loque americana es una de las enfermedades más devastadoras de la abeja melífera, causada por el bacilo, formador de esporas Grampositivo Paenibacillus larvae. El reciente ensamblaje y anotación del genoma de este patógeno permite actualmente la realización de profundos estudios moleculares. En este trabajo, se proporcionan técnicas y protocolos seleccionados para la investigación de la loque americana, principalmente bajo la forma de protocolos de trabajo con una estructura similar al de las recetas, para facilitar su implementación en el laboratorio. Los temas desarrollados incluyen: el trabajo con Paenibacillus larvae, técnicas básicas microbiológicas, la infección experimental, y "'ómicas" y otras técnicas sofisticadas. Además, este capítulo abarca otro tipo de información técnica, incluyendo medidas de bioseguridad para garantizar la seguridad en el manejo de este patógeno.

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