SummaryMethods 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. Métodos estándar para la investigación sobre Nosema ResumenSe describen procedimientos para trabajar con Nosema apis y Nosema ceranae en el campo y en el laboratorio. Para el trabajo de campo, se describen diferentes métodos de muestreo para determinar infecciones al nivel de colonia en un momento determinado, y también para el seguimiento de la dinámica temporal de infección. Se hacen sugerencias para la forma de estandarizar los ensayos de campo para evaluar los tratamientos y el impacto de la enfermedad. Los métodos de laboratorio descritos incluyen diferentes formas de determinar los niveles de infección al nivel de colonia y de abeja individual, y los métodos para la determinación de las especies, incluyendo microscopía óptica, microscopía electrónica y métodos moleculares (PCR). Se hacen sugerencias para estandarizar los ensayos con cajas, y se describen diferentes métodos de inoculación para infectar abejas, incluyendo métodos de control para la viabilidad de las esporas. Se describe un sistema de cultivo celular para la cría in vitro de Nosema spp. Finalmente, se describe cómo llevar a cabo diferentes tipos de experimentos, incluyendo la dosis infecciosa, efectos de la dosis, curso de la infección y las pruebas de longevidad.

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The frequency of occurrence and relative concentration of 44 pesticides in apicultural (Apis mellifera) matrices collected from five French locations (24 apiaries) were assessed from 2002 to 2005. The number and nature of the pesticides investigated varied with the matrices examined-living honeybees, pollen loads, honey, and beeswax. Pollen loads and beeswax had the highest frequency of pesticide occurrence among the apiary matrices examined in the present study, whereas honey samples had the lowest. The imidacloprid group and the fipronil group were detected in sufficient amounts in all matrices to allow statistical comparisons. Some seasonal variation was shown when residues were identified in pollen loads. Given the results (highest frequency of presence) and practical aspects (easy to collect; matrix with no turnover, unlike with bees that are naturally renewed), pollen loads were the best matrix for assessing the presence of pesticide residues in the environment in our given conditions.

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.

In 2002, a field survey was initiated on French apiaries to monitor weakness of honey bee, Apis mellifera L., colonies. Apiaries were evenly distributed in five sites located on continental France. Five colonies were randomly selected in each apiary, leading to a total of 125 studied honey bee colonies. For 3 yr (starting in autumn 2002), colonies were visited four times per year: after winter, before summer, during summer, and before winter. Pollen loads from traps were collected at each visit. Multiresidue analyses were performed in pollen to search residues of 36 different molecules. Specific analyses were conducted to search fipronil and metabolites and also imidacloprid and metabolites. Residues of 19 searched compounds were found in samples. Contamination by pesticides ranged from 50 to 0%. Coumaphos and tau-fluvalinate residues were the most concentrated of all residues (mean concentrations were 925.0 and 487.2 microg/kg, respectively). Fipronil and metabolite contents were superior to the limit of detection in 16 samples. Residues of fipronil were found in 10 samples. Nine samples contained the sulfone compound, and three samples contained the desulfinyl compound. Residues of imidacloprid and 6-chloronicotinic acid were found in 69% of samples. Imidacloprid contents were quantified in 11 samples with values ranging from 1.1 to 5.7 microg/kg. 6-Chloronicotinic acid content was superior to the limit of quantification in 28 samples with values ranging from 0.6 to 9.3 microg/kg. Statistical tests showed no difference between places of sampling with the exception of fipronil. Possible origins of these contaminations, concentration and toxicity of pesticides, and the possible consequences for bees are discussed.

A 3-yr field survey was carried out in France, from 2002 to 2005, to study honey bee (Apis mellifera L.) colony health in relation to pesticide residues found in the colonies. This study was motivated by recent massive losses of honey bee colonies, and our objective was to examine the possible relationship between low levels of pesticide residues in apicultural matrices (honey, pollen collected by honey bees, beeswax) and colony health as measured by colony mortality and adult and brood population abundance. When all apicultural matrices were pooled together, the number of pesticide residue detected per sampling period (four sampling periods per year) and per apiary ranged from 0 to 9, with the most frequent being two (29.6%). No pesticide residues were detected during 12.7% of the sampling periods. Residues of imidacloprid and 6- chloronicotinic acid were the most frequently detected in pollen loads, honey, and honey bee matrices. Several pairs of active ingredients were present concurrently within honey bees and in pollen loads but not in beeswax and honey samples. No statistical relationship was found between colony mortality and pesticide residues. When pesticide residues from all matrices were pooled together, a mixed model analysis did not show a significant relationship between the presence of pesticide residues and the abundance of brood and adults, and no statistical relationship was found between colony mortality and pesticide residues. Thus, although certain pesticide residues were detected in apicultural matrices and occasionally with another pesticide residual, more work is needed to determine the role these residues play in affecting colony health.

Over the last few years, many European and North American countries have reported a high rate of disorders (mortality, dwindling and disappearance) affecting honeybee colonies (Apis mellifera). Although beekeeping has become an increasingly professional activity in recent years, the beekeeping industry remains poorly documented in Europe. The European Union Reference Laboratory for Honeybee Health sent a detailed questionnaire to each Member State, in addition to Kosovo and Norway, to determine the demographics and state of their beekeeping industries. Based on data supplied by the National Reference Laboratory for honeybee diseases in each European country, a European database was created to describe the beekeeping industry including the number and types of beekeepers, operation size, industry production, and health (notifiable diseases, mortalities). The total number of beekeepers in Europe was estimated at 620 000. European honey production was evaluated at around 220 000 tons in 2010. The price of honey varied from 1.5 to 40 €/kg depending on the country and on the distribution network. The estimated colony winter mortality varied from 7 to 28% depending on the country and the origin of the data (institutional survey or beekeeping associations). This survey documents the high heterogeneity of the apicultural industry within the European Union. The high proportion of non-professional beekeepers and the small mean number of colonies per beekeeper were the only common characteristics at European level. The tremendous variation in European apicultural industries has implication for any comprehensive epidemiological or economic analysis of the industry. This variability needs to be taken into account for such analysis as well as for future policy development. The industry would be served if beekeeping registration was uniformly implemented across member states. Better information on the package bee and queen production would help in understanding the ability of the industry to replace lost honey bee stocks.

In 2002 a field survey was initiated in French apiaries in order to monitor the health of honey bee colonies (Apis mellifera L.). Studied apiaries were evenly distributed across five sites located in continental France. Beeswax samples were collected once a year over 2 years from a total of 125 honey bee colonies. Multiresidue analyses were performed on these samples in order to identify residues of 16 insecticides and acaricides and two fungicides. Residues of 14 of the searched-for compounds were found in samples. Tau-fluvalinate, coumaphos and endosulfan residues were the most frequently occurring residues (61.9, 52.2 and 23.4% of samples respectively). Coumaphos was found in the highest average quantities (792.6 microg kg(-1)). Residues of cypermethrin, lindane and deltamethrin were found in 21.9, 4.3 and 2.4% of samples respectively. Statistical tests showed no difference between years of sampling, with the exception of the frequency of pyrethroid residues. Beeswax contamination was the result of both in-hive acaricide treatments and, to a much lesser extent, environmental pollution.