| 1. |
- Andonov, Sreten
(author)
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Standard methods for direct observation of honey bee (Apis mellifera L.) nuptial flights
- 2024
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In: Journal of Apicultural Research. - 0021-8839 .- 2078-6913. ; 63, s. 65-75
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Journal article (peer-reviewed)abstract
- Honey bees (Apis mellifera) have a peculiar and complex reproductive biology, with queens being polyandrous and mating with several drones during one or more mating (nuptial) flights in so-called drone congregation areas. Observing the virgin queens' and drones' flight behaviour provides data to understand and interpret a portion of the honey bees' complex reproductive process. Observing the behaviour of the virgin queens on the hive entrance also serves to estimate the distance from the mating place or potential drone congregation areas (DCAs) as well as to detect the presence of airborne drones in the area.In this paper, we provide a detailed description of the methodology used for observing queens' and drones' flights during the period of expected mating. In addition, we provide information about required equipment, tools as well as step by step description of the observation and recordkeeping process.
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| 2. |
- Diaz Calafat, Joan
(author)
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The Asian giant resin bee Megachile sculpturalis Smith 1853 (Hymenoptera: Apoidea: Megachilidae), a new exotic species for the bee fauna of Mallorca (Balearic Islands, Spain)
- 2021
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In: Journal of Apicultural Research. - : Informa UK Limited. - 0021-8839 .- 2078-6913. ; 60, s. 506-511
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Journal article (peer-reviewed)abstract
- Megachile sculpturalis Smith 1853 is found for the first time on the island of Mallorca (Balearic Islands, Spain). This is the first time that this exotic bee is reported from an island and is the southernmost record in Europe so far. Specimens were found in five distant locations throughout the island, which points out the current broad distribution of this species within the island and suggests that the introduction of M. sculpturalis may have happened earlier than 2020 and went unnoticed. M. sculpturalis females were found mainly feeding and collecting pollen from Sophora japonica L. 1767 (= Styphnolobium japonicum (L.) Schott 1830) and Parkinsonia aculeata L. 1753, two exotic ornamental trees. Potential entry means and impacts of this exotic bee on local biodiversity are discussed. Island ecosystems are highly vulnerable to the negative impacts produced by exotic species. Judging from the evidence of negative effects described in the literature so far, we recommend that the invasive potential and the impact of this species on native plants and pollinators within the insular context of the Balearic Islands be addressed in the future.
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| 3. |
- Fabricius Kristiansen, Lotta
(author)
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COLOSS survey: global impact of COVID-19 on bee research
- 2020
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In: Journal of Apicultural Research. - : Informa UK Limited. - 0021-8839 .- 2078-6913. ; 59, s. 731-734
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Journal article (peer-reviewed)abstract
- The socio-economic impacts of COVID-19 on society have yet to be truly revealed; there is no doubt that the pandemic has severely affected the daily lives of most of humanity. It is to be expected that the research activities of scientists could be impacted to varying degrees, but no data exist on how COVID-19 has affected research specifically. Here, we show that the still ongoing COVID-19 pandemic has already diversely and negatively affected bee research at a global level. An online survey disseminated through the global COLOSS honey bee research association showed that every participant (n = 230 from 56 countries) reported an impact on one or more of their activities. Activities that require travelling or the physical presence of people (meetings and conferences, teaching and extension) were affected the most, but also laboratory and field activities, daily operations, supervision and other activities were affected to varying degrees. Since the basic activities are very similar for many research fields, it appears as if our findings for bee research can be extrapolated to other fields. In the light of our data, we recommend that stakeholders such as governments and funding bodies who support research should facilitate the wide implementation of web-based information technology required for efficient online communication for research and education, as well as adequately loosened restriction measures with respect to field and laboratory work. Finally, increased flexibility in administration and extension of research grants and fellowships seem to be needed. It is apparent that adequate responses by all stakeholders are required to limit the impact of COVID-19 and future pandemics on bee science and other research fields.
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| 4. |
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| 5. |
- Forsgren, Eva
(author)
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First detection of Nosema ceranae in New Zealand honey bees
- 2015
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In: Journal of Apicultural Research. - : Informa UK Limited. - 0021-8839 .- 2078-6913. ; 54, s. 358-365
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Journal article (peer-reviewed)abstract
- Nosema is a genus of 81 microsporidian parasites, two of which affect honey bees. The advent of molecular methods has facilitated the differentiation between Nosema apis and Nosema ceranae. N. ceranae infection was detected by polymerase chain reaction (PCR) in an apiary on the Coromandel Peninsula of New Zealand during investigation into unusual colony losses in early spring 2010, and was subsequently found in other areas of the country. While no association could be confirmed between apiary health status and presence of N. ceranae, a significantly higher proportion of positive apiaries were detected in spring compared with autumn. PCR for N. ceranae conducted in three different laboratories showed fair agreement. Minor assay differences and sample level variation in Nosema are the most likely explanation for differences in results.
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| 6. |
- Forsgren, Eva, et al.
(author)
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Miscellaneous standard methods for Apis mellifera research
- 2013
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In: Journal of Apicultural Research. - 0021-8839 .- 2078-6913. ; 52, s. 1-55
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Research review (peer-reviewed)abstract
- A variety of methods are used in honey bee research and differ depending on the level at which the research is conducted. On an individual level, the handling of individual honey bees, including the queen, larvae and pupae are required. There are different methods for the immobilising, killing and storing as well as determining individual weight of bees. The precise timing of developmental stages is also an important aspect of sampling individuals for experiments. In order to investigate and manipulate functional processes in honey bees, e. g. memory formation and retrieval and gene expression, microinjection is often used. A method that is used by both researchers and beekeepers is the marking of queens that serves not only to help to locate her during her life, but also enables the dating of queens. Creating multiple queen colonies allows the beekeeper to maintain spare queens, increase brood production or ask questions related to reproduction. On colony level, very useful techniques are the measurement of intra hive mortality using dead bee traps, weighing of full hives, collecting pollen and nectar, and digital monitoring of brood development via location recognition. At the population level, estimation of population density is essential to evaluate the health status and using beelines help to locate wild colonies. These methods, described in this paper, are especially valuable when investigating the effects of pesticide applications, environmental pollution and diseases on colony survival.
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| 7. |
- Forsgren, Eva
(author)
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Standard methods for European foulbrood research
- 2013
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In: Journal of Apicultural Research. - 0021-8839 .- 2078-6913. ; 52, s. 1-14
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Research review (peer-reviewed)abstract
- European foulbrood (EFB) is a severe bacterial honey bee brood disease caused by the Gram-positive bacterium Melissocccus plutonius. The disease is widely distributed worldwide, and is an increasing problem in some areas. Although the causative agent of EFB was described almost a century ago, many basic aspects of its pathogenesis are still unknown. Earlier studies were hampered by insensitive and unspecific methods such as culture based techniques. Recent advances in molecular technology are making it increasingly easy to detect and characterize microbes, and nucleic acid detection technologies are quickly displacing the traditional phenotypic assays in microbiology. This paper presents selected methodologies which focus on EFB and its causative agent M. plutonius.
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| 8. |
- Fries, Ingemar
(author)
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Standard methods for maintaining adult Apis mellifera in cages under in vitro laboratory conditions
- 2013
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In: Journal of Apicultural Research. - 0021-8839 .- 2078-6913. ; 52
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Research review (peer-reviewed)abstract
- Adult honey bees are maintained in vitro in laboratory cages for a variety of purposes. For example, researchers may wish to perform experiments on honey bees caged individually or in groups to study aspects of parasitology, toxicology, or physiology under highly controlled conditions, or they may cage whole frames to obtain newly emerged workers of known age cohorts. Regardless of purpose, researchers must manage a number of variables, ranging from selection of study subjects (e. g. honey bee subspecies) to experimental environment (e. g. temperature and relative humidity). Although decisions made by researchers may not necessarily jeopardize the scientific rigour of an experiment, they may profoundly affect results, and may make comparisons with similar, but independent, studies difficult. Focusing primarily on workers, we provide recommendations for maintaining adults under in vitro laboratory conditions, whilst acknowledging gaps in our understanding that require further attention. We specifically describe how to properly obtain honey bees, and how to choose appropriate cages, incubator conditions, and food to obtain biologically relevant and comparable experimental results. Additionally, we provide broad recommendations for experimental design and statistical analyses of data that arises from experiments using caged honey bees. The ultimate goal of this, and of all COLOSS BEEBOOK papers, is not to stifle science with restrictions, but rather to provide researchers with the appropriate tools to generate comparable data that will build upon our current understanding of honey bees.
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| 9. |
- Fries, Ingemar
(author)
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Standard methods for Nosema research
- 2013
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In: Journal of Apicultural Research. - 0021-8839 .- 2078-6913. ; 52
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Research review (peer-reviewed)abstract
- Methods are described for working with Nosema apis and Nosema ceranae in the field and in the laboratory. For fieldwork, different sampling methods are described to determine colony level infections at a given point in time, but also for following the temporal infection dynamics. Suggestions are made for how to standardise field trials for evaluating treatments and disease impact. The laboratory methods described include different means for determining colony level and individual bee infection levels and methods for species determination, including light microscopy, electron microscopy, and molecular methods (PCR). Suggestions are made for how to standardise cage trials, and different inoculation methods for infecting bees are described, including control methods for spore viability. A cell culture system for in vitro rearing of Nosema spp. is described. Finally, how to conduct different types of experiments are described, including infectious dose, dose effects, course of infection and longevity tests.
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| 10. |
- Lamei, Sepideh, et al.
(author)
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The secretome of honey bee-specific lactic acid bacteria inhibits Paenibacillus larvae growth
- 2019
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In: Journal of Apicultural Research. - : Informa UK Limited. - 0021-8839 .- 2078-6913. ; 58:3, s. 405-412
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Journal article (peer-reviewed)abstract
- American Foulbrood (AFB) is a particularly pernicious bacterial disease of honey bees due to the extreme persistence of endospores of the causative agent Paenibacillus larvae. These spores are resistant to harsh environmental conditions, unaffected by antimicrobial agents and can remain viable for decades. The germination of the endospore in the larval midgut is the crucial first step leading to infection, followed by vegetative growth, tissue invasion and disease, culminating in spore formation when the host´s nutrients have been exhausted. Therefore, inhibiting spore germination or impeding early vegetative growth would be a highly effective strategy for limiting the impact of AFB. We previously showed that honey bee-specific lactic acid bacteria (hbs–LAB) had a major inhibitory effect on P. larvae both in culture and in larval bioassays. The present study documents the progress towards characterization of compounds, processes and interactions between P. larvae and the hbs–LAB responsible for this inhibitory effect. Firstly, we used an agar diffusion assay and larval infection bioassay to show that most, if not all, of the inhibitory effect was associated with the extracellular fraction (secretome). Secondly, we employed a turbidimetric growth assay to demonstrate that the hbs–LAB secretome strongly inhibited P. larvae vegetative growth, however, probably not by reducing spore germination. The inhibition was similarly effective against both major P. larvae genotypes (ERIC-I and II) in all experiments. The implications of our results for characterization of the secretome and for the management and treatment of AFB and P. larvae are further discussed.
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| 11. |
- Locke Grandér, Barbara
(author)
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Standard methods for varroa research
- 2013
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In: Journal of Apicultural Research. - 0021-8839 .- 2078-6913. ; 52, s. 1-54
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Journal article (peer-reviewed)abstract
- Very rapidly after Varroa destructor invaded apiaries of Apis mellifera, the devastating effect of this mite prompted an active research effort to understand and control this parasite. Over a few decades, varroa has spread to most countries exploiting A. mellifera. As a consequence, a large number of teams have worked with this organism, developing a diversity of research methods. Often different approaches have been followed to achieve the same goal. The diversity of methods made the results difficult to compare, thus hindering our understanding of this parasite. In this paper, we provide easy to use protocols for the collection, identification, diagnosis, rearing, breeding, marking and measurement of infestation rates and fertility of V. destructor. We also describe experimental protocols to study orientation and feeding of the mite, to infest colonies or cells and measure the mite's susceptibility to acaricides. Where relevant, we describe which mite should be used for bioassays since their behaviour is influenced by their physiological state. We also give a method to determine the damage threshold above which varroa damages colonies. This tool is fundamental to be able to implement integrated control concepts. We have described pros and cons for all methods for the user to know which method to use under which circumstances. These methods could be embraced as standards by the community when designing and performing research on V. destructor.
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| 12. |
- Mazi, S., et al.
(author)
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A subset of wild bee species boosts the pollination of pigeon pea (Cajanus cajan: Fabaceae), an important crop plant of Cameroon
- 2022
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In: Journal of Apicultural Research. - : Informa UK Limited. - 0021-8839 .- 2078-6913. ; 62:3, s. 598-606
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Journal article (peer-reviewed)abstract
- Bee species are thought to vary in their pollination efficiency, but they are rarely compared, particularly in the tropics. Here we determined the role in the pollination of 13 native bee species (Apis mellifera and 12 other wild bee species) when visiting pigeon pea (Cajanus cajan) flowers across two growing seasons in Cameroon. Using observations of floral visits coupled with a field experiment to quantify single-visit pollination efficiency, we found that Chalicodoma rufipes was the most efficient pollinator and most abundant flower visitor of pigeon pea. Most other flower visitors, including Apis mellifera, detracted from pigeon pea seed set. Our study highlights the importance of quantifying pollination to reveal functionally important bee species.
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| 13. |
- Nilsson, Anna, et al.
(author)
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Honey bee pathogens and parasites in Swedish apiaries: a baseline study
- 2021
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In: Journal of Apicultural Research. - : Informa UK Limited. - 0021-8839 .- 2078-6913. ; 60, s. 697-706
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Journal article (peer-reviewed)abstract
- This nation-wide survey including 382 Swedish apiaries is the first to document base-line information of the prevalence and distribution of the ectoparasitic mite Varroa destructor, the mite-associated viruses Deformed wing virus and Acute bee paralysis virus, and the bacterial honey bee pathogens Paenibacillus larvae and Melissococcus plutonius in the country. Varroa and associated viruses were not detected in the northern regions of the country. The bacterium P. larvae was detected in 6% of the investigated apiaries and it was absent in more than half of the counties, M. plutonius was detected in two apiaries in one county. Other results from our study include questionnaire responses, in which beekeepers reported total winter colony losses of 6.4%. Fifty-three percent of the beekeepers reported to have purchased queens the year preceding this study, and 40.1% moved colonies to the apiary. Queens were imported from European countries and the USA. The movements of bees were one of the factors strongly associated with the prevalence of the disease-causing organisms surveyed and colony losses. The majority of the participating beekeepers were not aware of any disease related signs in their apiaries despite positive laboratory findings. This highlights the importance of further outreach efforts to increase the beekeepers' awareness of diseases and disease management. The results provide a disease baseline for improvements of the surveillance system.
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| 14. |
- Perichon, Samuel, et al.
(author)
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Advice on how to start beekeeping, memories with bees and the uses of honey : results of an online questionnaire with European beekeepers
- 2024
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In: Journal of Apicultural Research. - : Taylor & Francis Group. - 0021-8839 .- 2078-6913. ; , s. 1-16
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Journal article (peer-reviewed)abstract
- Beekeeping is a demanding activity that requires both particular human qualities from those who practise it and an environment that is favourable to bees. This is why the interviewed beekeepers advise to take time to think before starting to keep the bees, to find a mentor, and to always have the desire to get to know the bees even if this is not enough for success. In Northern and Western Europe, beekeepers consider patience and calmness as essential qualities to be a “good beekeeper”, while in Southern Europe, passion for bees is the main driver of success. The reasons for abandonment or failure also include human and environmental factors. To better understand the relationship between beekeepers and their bees, interviewees were encouraged to share their best memories. With years of experience, contemplative memories fade into memorable situations in which bee practices are described, and then in turn, they fade into moments of sociability. Honey harvesting plays an important role. Productive considerations are more expressed in Southern Europe, where it is an exceptional harvest that marks on people’s minds, more than the first honey harvest. The strong connection to their bees and the territory they live in is also expressed in the choice of their favourite honey. Many beekeepers mentioned the local honey production, which they are proud to produce with their own bees, even though they sometimes became criticized for a too high price for this local product.
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| 15. |
- Potts, Simon G., et al.
(author)
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Developing European conservation and mitigation tools for pollination services: approaches of the STEP (Status and Trends of European Pollinators) project
- 2011
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In: Journal of Apicultural Research. - 0021-8839 .- 2078-6913. ; 50:2, s. 152-164
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Research review (peer-reviewed)abstract
- Pollinating insects form a key component of European biodiversity, and provide a vital ecosystem service to crops and wild plants. There is growing evidence of declines in both wild and domesticated pollinators, and parallel declines in plants relying upon them. The STEP project (Status and Trends of European Pollinators, 2010-2015, www.step-project.net) is documenting critical elements in the nature and extent of these declines, examining key functional traits associated with pollination deficits, and developing a Red List for some European pollinator groups. Together these activities are laying the groundwork for future pollinator monitoring programmes. STEP is also assessing the relative importance of potential drivers of pollinator declines, including climate change, habitat loss and fragmentation, agrochemicals, pathogens, alien species, light pollution, and their interactions. We are measuring the ecological and economic impacts of declining pollinator services and floral resources, including effects on wild plant populations, crop production and human nutrition. STEP is reviewing existing and potential mitigation options, and providing novel tests of their effectiveness across Europe. Our work is building upon existing and newly developed datasets and models, complemented by spatially-replicated campaigns of field research to fill gaps in current knowledge. Findings are being integrated into a policy-relevant framework to create evidence-based decision support tools. STEP is establishing communication links to a wide range of stakeholders across Europe and beyond, including policy makers, beekeepers, farmers, academics and the general public. Taken together, the STEP research programme aims to improve our understanding of the nature, causes, consequences and potential mitigation of declines in pollination services at local, national, continental and global scales.
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| 16. |
- Rodrigues De Miranda, Joachim
(author)
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Standard methods for molecular research in Apis mellifera
- 2013
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In: Journal of Apicultural Research. - 0021-8839 .- 2078-6913. ; 52
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Journal article (peer-reviewed)abstract
- 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.
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| 17. |
- Rodrigues De Miranda, Joachim
(author)
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Standard methods for virus research in Apis mellifera
- 2013
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In: Journal of Apicultural Research. - 0021-8839 .- 2078-6913. ; 52
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Journal article (peer-reviewed)abstract
- Honey 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.
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| 18. |
- Rodrigues De Miranda, Joachim
(author)
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Statistical guidelines for Apis mellifera research
- 2013
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In: Journal of Apicultural Research. - 0021-8839 .- 2078-6913. ; 52
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Journal article (peer-reviewed)abstract
- In this article we provide guidelines on statistical design and analysis of data for all kinds of honey bee research. Guidelines and selection of different methods presented are, at least partly, based on experience. This article can be used: to identify the most suitable analysis for the type of data collected; to optimise one's experimental design based on the experimental factors to be investigated, samples to be analysed, and the type of data produced; to determine how, where, and when to sample bees from colonies; or just to inspire. Also included are guidelines on presentation and reporting of data, as well as where to find help and which types of software could be useful.
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| 19. |
- Rodrigues De Miranda, Joachim, et al.
(author)
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Varroa destructor : Research avenues towards sustainable control.
- 2012
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In: Journal of Apicultural Research. - 0021-8839 .- 2078-6913. ; 51, s. 125-132
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Research review (peer-reviewed)abstract
- Pollination by honey bees plays a key role in the functioning of ecosystems and optimisation of agricultural yields. Severe honey bee colony losses worldwide have raised concerns about the sustainability of these pollination services. In many cases, bee mortality appears to be the product of many interacting factors, but there is a growing consensus that the ectoparasitic mite Varroa destructor plays the role of the major predisposing liability. We argue that the fight against this mite should be a priority for future honey bee health research. We highlight the lack of efficient control methods currently available against the parasite and discuss the need for new approaches. Gaps in our knowledge of the biology and epidemiology of the mite are identified and a research road map towards sustainable control is drawn. Innovative and challenging approaches are suggested in order to stimulate research efforts and ensure that honey bees will be able to sustainably fulfil their role in the ecosystem.
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| 20. |
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| 21. |
- Olofsson, Tobias, et al.
(author)
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Phylogenetic comparison of bacteria isolated from the honey stomachs of honey bees Apis mellifera and bumble bees Bombus spp.
- 2009
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In: Journal of Apicultural Research. - 0021-8839. ; 48:4, s. 233-237
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Journal article (peer-reviewed)abstract
- It has recently been discovered that the honey bee Apis mellifera has a large flora of symbiotic lactic acid bacteria in its honey stomach, belonging to the genera Lactobacillus and Bifidobacterium. It appears that the flora may protect the honey bees, their larvae and their food against harmful microorganisms. Since bumble bees (Bombus spp.) are related to honey bees and have a honey stomach where they store nectar during their flight we investigated whether their honey stomachs also contain symbiotic lactic acid bacteria. Bacterial isolates cultivated from both the surface and from the honey stomachs of bumble bees were identified using 16S rRNA gene analyzes. The results showed that bumble bees also possess lactic acid bacteria in their honey stomachs but in fewer phylotypes and only belonging to the genus Lactobacillus In contrast to honey bees, bumble bees do not produce honey or bee bread but feed their larvae directly with nectar and pollen, and their society does not survive the winter in temperate climates. It was therefore concluded that bumble bees have less need than honey bees of an extensive lactic acid bacterial flora.
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| 22. |
- Vasquez, Alejandra, et al.
(author)
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The lactic acid bacteria involved in the production of bee pollen and bee bread
- 2009
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In: Journal of Apicultural Research. - 0021-8839. ; 48:3, s. 189-195
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Journal article (peer-reviewed)abstract
- Recently a large flora of lactic acid bacteria (LAB) was identified in the honey stomach of the honey bee Apis mellifera. In this study, the presence of this flora in bee pollen and bee bread was investigated. Pollen was collected from the legs of honey bees, and both two week old and two month old bee bread were also obtained for the study. Bacterial isolates cultivated from these bee products were identified using 16S rRNA gene analyzes. The majority of the honey stomach LAB flora was recovered in a viable state from both the pollen and the two week old bee bread, but not from the two month old bee bread. It is demonstrated for the first time that bee bread is probably fermented by the honey stomach LAB flora that has been added to the pollen via regurgitated nectar from the honey stomach. This discovery helps to explain how honey bees standardize the production of bee bread and how it is stored. The presence of the honey stomach LAB and its antimicrobial substances in bee bread also suggests a possible role in the defence against honey bee diseases since the bee bread is consumed by both the larvae and the adult bees.
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| 23. |
- Nilsson, Anna, et al.
(author)
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Cutinase-peptide fusions in thermoseparating aqueous two-phase systems - Prediction of partitioning and enhanced tag efficiency by detergent addition
- 2002
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In: Journal of Chromatography A. - 0021-9673. ; 946:1-2, s. 141-155
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Journal article (peer-reviewed)abstract
- It is of increasing importance to develop efficient purification methods for recombinant proteins where the number of steps can be minimised. The aim has been to establish a method for predicting the partitioning of the wild-type target protein in an aqueous two-phase system, and with this as basis, develop fusion tags and optimise the phase system for enhanced partitioning of the target protein. The surface of the lipolytic enzyme cutinase from Fusarium solani pisi was investigated with a computer program, Graphical Representation and Analysis of Surface Properties (GRASP). The accessible surface areas for the different amino acid residues were used together with peptide partitioning data to calculate the partition coefficient for the protein. The separation system was composed of a thermoseparating random copolymer of ethylene oxide and propylene oxide, Breox PAG 50A 1000, as top phase forming polymer and a hydroxypropyl starch polymer, Reppal PES 200, as bottom phase polymer. The calculated partition coefficient for the wild-type protein (K=1.0) agreed reasonably well with the experimentally determined value (K=0.85). Genetic engineering was used to construct fusion proteins expressed in Saccharomyces cerevisiae based on cutinase and peptide tags containing tryptophan, to enhance the partitioning in aqueous two-phase systems. The partitioning of the cutinase constructs could qualitatively be predicted from peptide partitioning data, i.e. the trends in partitioning could be predicted. A spacer peptide introduced between protein and tag increased the partitioning of the protein towards the ethylene oxide-propylene oxide (EOPO) copolymer top phase. The aqueous two-phase system was modified by addition of detergent to increase the partitioning of the cutinase variants towards the EOPO copolymer phase. Triton and a series of C12En detergents selectively increased the partitioning of cutinase constructs with (WP)4-based tags up to 14 times compared to wild-type cutinase. The protein partition could almost quantitatively be predicted from the peptide partition data.
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| 24. |
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