EurBee board Dorothea Brückner, Germany Norberto Milani, Italy Robert Paxton, Great Britain Dalibor Titěra, Czech Republic Bernard Vaissiere, France Program consultant
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Can honeybees learn by example? Lars Chittka, Ellouise Leadbeater, Nigel Raine School of Biological and Chemical Sciences, Queen Mary, University of London, Mile End Road, London E1 4NS, UK Social learning in insects has received much less attention in insects than in vertebrates, but the literature contains several intriguing examples. Darwin suggested that honeybees can copy from bumblebees how to rob nectar from long-spurred flowers, by observatory learning. Indeed, new research indicates that there might be cases of social learning in flower-visiting bees. Recent publications even claim that teaching might occur in some social insects. Could the honeybee dance be a case that contains both observatory learning by recruits and teaching by dancers? We discuss the evidence and possible experiments, including the feedback from recruits to dancers, that would be necessary to answer these questions.
Centre de Recherches sur la Cognition Animale (UMR 5169), CNRS - Université Paul Sabatier, 118 route de Narbonne, 31062 Toulouse cedex 4, France., E-mail: benard©cict.fr Categorization refers to the classification of perceptual input into defined functional groups. We present and discuss evidence suggesting that stimulus categorization can also be found in an invertebrate, the honeybee Apis mellifera, thus underlining the generality across species of this cognitive process. Honeybees show positive transfer of appropriate responding from a trained to a novel set of visual stimuli. Such a transfer was demonstrated for specific isolated features such as symmetry or orientation, but also for topographic layouts of features. Such assemblies may involve different orientations of a single element (e.g., a bar), or even different elements building structured or unstructured face-like stimuli. Moreover, bees could learn very well to categorize short- versus long-wavelength stimuli (as they discriminate between and generalized within categories) and they generalized successfully their choice to new colors belonging to these categories. Though in most cases specific experimental controls such as stimulus balance and discriminability are still required, it seems appropriate to characterize the performance of honeybees as reflecting categorization. Further experiments should address the issue of which categorization model accounts better for the visual performances of honeybees.
Department of Biological Sciences, Graduate School of Science, The University of Tokyo, Bunkyo-ku, Tokyo 113-0033, Japan, E-mail: takeuchi©biol.s.u-tokyo.ac.jp The honeybee is a social insect and its colony consists of a queen, workers and drones that have different roles. In addition, various complex social behaviors, such as communications and divisions of labor, are performed by colony members to maintain colony activities. Mushroom body (MB) is one of insect brain structures, which is important for learning, memory and sensory integration. Honeybee MB function is believed to be closely associated with honeybee social behavior for the following reasons: 1) The MBs of the aculeate Hymenoptera, including the honeybee, are more prominent compared with those of other insects. 2) Honeybee MBs have a high degree of structural plasticity and the volume of the neuropil varies according to the division of labor. To clarify molecular basis underlying honeybee MBs function, we identified over 20 genes expressed selectively in the MBs of the honeybee brain using combination of differential display and cDNA microarray method. In this talk, I will present the expression pattern of these MB-selective genes and discuss possible function of these genes in comparison with those of their orthologues in non-social insects/invertabrate. We also study about visual learning and chromatic adaptation in the harnessed honeybee and will examine involvement of these genes in the honeybee visual behaviors. Reference Hori S, Takeuchi H, et al. (2006) Associative visual learning, color discrimination, and chromatic adaptation in the harnessed honeybee. Journal of Comparative Physiology A., in press. Associative visual learning, color discrimination, and chromatic adaptation in the harnessed honeybee Sayaka Hori1, Hideaki Takeuchi1, Kentaro Arikawa2, Michiyo Kinoshita2, Naoko Ichikawa3, Masami Sasaki3, Takeo Kubo1 1;Department of Biological Sciences, Graduate School of Science, The University of Tokyo 2; Laboratory of Neuroethology, The Graduate University for Advanced Studies, 3; Faculty of Agriculture, Tamagawa University, E-mail: holis©biol.s.u-tokyo.ac.jp To analyze visual cognitive capacity in the honeybee, we studied associative visual learning in harnessed honeybees trained with monochromatic lights associated with a sucrose solution, to elicit the proboscis extension reflex (PER). We clarified five properties of visual learning. 1, Antennae deprivation significantly increased the learning rate, suggesting that sensory input from the antennae interferes with the light-PER associative learning in this conditioning paradigm. 2, Shading the compound eyes with silver paste significantly decreased the learning rate, while shading the ocelli did not. These results indicate that the visual information received by the compound eyes was crucial in this conditioning paradigm. 3, There was no significant difference in the visual learning between nurse bees, guard bees, and forager bees. These results indicated that these workers have almost equivalent ability for light-PER associative learning. 4, Bees conditioned with 540nm light stimulus exhibited light-induced PER with 618nm, but not with 439nm light stimulus. These results indicate that the 540nm light-conditioned bees generalized between 618nm and 540nm light stimuli, whereas they did not generalize between 540nm and 439nm. 5, Bees conditioned with 540nm light stimulus exhibited PER immediately after the 439nm light was turned off, suggesting that the bees reacted to an afterimage induced by prior adaptation to the 439nm light, which might be similar to 540nm light.
The University of Tokyo, E-mail: holis©biol.s.u-tokyo.ac.jp To analyze visual cognitive capacity in the honeybee, we studied associative visual learning in harnessed honeybees trained with monochromatic lights associated with a reward of sucrose solution delivered to the proboscis, to elicit the proboscis extension reflex (PER). We clarified five properties of visual learning under these conditions. First, antennae deprivation significantly increased the learning rate. These results suggested that sensory input from the antennae (olfactory and/or mechanical stimulation) interferes with the light-PER associative learning in this conditioning paradigm. Second, shading the compound eyes with silver paste significantly decreased the learning rate, while shading the ocelli did not. These results indicate that the visual information received by the compound eyes was crucial in this conditioning paradigm. Third, there was no significant difference in the visual learning between nurse bees, guard bees, and forager bees. These results indicated that these workers have almost equivalent ability for light-PER associative learning, independent of their roles in the colony. Forth, bees conditioned with 540nm light stimulus exhibited light-induced PER with 618nm, but not with 439nm light stimulus. These results indicate that the 540nm light-conditioned bees generalized between 618nm and 540nm light stimuli, whereas they did not generalize between 540nm and 439nm. Finally, bees conditioned with 540nm light stimulus exhibited PER immediately after the 439nm light was turned off, suggesting that the bees reacted to an afterimage induced by prior adaptation to the 439nm light, which might be similar to 540nm light. A combination of behavioral and neurobiological methods will contribute to clarify the neural circuit and molecular mechanisms underlying visual processing in honeybees.
PPO Bee Unit, Wageningen University & Research, Wageningen,The Netherlands E-mail: tjeerd.blacquiere©wur.nl New greenhouse cladding materials are developed with the aim to reduce the loss of heat from the greenhouse, and simultaneously to maximize the transmission of light for plant growth (wavelengths 400-700 nm). The new claddings are made of polymers, with transmission characteristics differing from those of glass, and double layered, while glass is only one layer. How do honeybees behave and forage under these conditions? The signals and information they normally use are absent or at least altered: reduced sight on the position of the sun, loss of polarization of light that has passed at least four times a gas-solid boundary and loss of ultra-violet radiation. In addition a greenhouse is in general as such a difficult surrounding for bees because of the lack of landscape variation, the use of screens that turn normal light distribution upside down and behind which bees easily get confused and lost, and vents with or without insect gauze. On a nursery with compartments with different claddings the orientation behaviour of worker bees of newly introduced colonies in small hives (MiniBeuten) was registered. Counts were made of the number of bees that went out for an orientation flight and of the numbers that returned to the hive. The claddings were glass, polymethylmethacrylate (PMMA) and polycarbonate (PC). Honey bees leaving the hive behaved normal under glass and PMMA, by flying in circles increasing in width around the hive, but divergent under PC, where they almost immediately went up and with a few loops ended in the top of the greenhouse on the insect gauze of the vents. Beneath glass and PMMC many bees returned to the hives, beneath PC hardly any bee returned. Bumblebees experienced similar problems in the PC greenhouse. Physiology and Behaviour Symposium organized by Martin Giurfa/Lars Chittka Differences between foraging groups in a learning task but no correlation in performance between tasks Sharoni Shafir1, Tamar Drezner-Levy1, Brian H. Smith2 1B. Triwaks Bee Research Center, Department of Entomology, Faculty of Agricultural, Food and Environmental Quality Sciences, The Hebrew University of Jerusalem, Rehovot, Israel. 2 School of Life Sciences, Arizona State University, P.O. Box 874501, Tempe, AZ 85287-4501, USA.
Due to multiple matings by the queen, a honey bee colony consists of foragers from several patrilines. These partrilines are known to differ in their tendency to collect pollen or nectar, and in various learning tasks. We tested returning foragers in a simple learning task by conditioning of the proboscis-extension response (PER). Subjects received three acquisition trials in which an odor was associated with a sucrose reward. Bees that had foraged for pollen and water learned better than those that foraged for nectar or for both nectar and pollen. Subjects were then tested in a PER assay modified for simultaneous choice between two odors, for their preference between an odor associated with a constant reward and another associated with a variable reward. Bees were risk averse in that they preferred the constant alternative, but levels of risk aversion did not differ between the various foraging groups. Finally, subjects were tested for latent inhibition. They experienced 30 unrewarded trials of exposure to an odor, followed by six acquisition trials with that odor. Learning of the inhibiting odor during the inhibition phase was manifest by retarded learning during the acquisition phase for the latent inhibition group relative to a control group, but there were no differences between the foraging groups. Our results suggest that different genes govern foraging specialization and performance in various learning tasks. Do honeybees, Apis mellifera scutellata, regulate humidity in their nest? Hannelie Human , Sue W. Nicolson, Vincent Dietemann University of Pretoria, E-mail: hhuman©zoology.up.ac.za Honeybee colonies are highly efficient at regulating the biophysical parameters of their hive. Constant temperature is crucial for the normal growth of the immature stages, and temperature regulation has been studied extensively. Humidity is also essential for brood development as well as for osmotic balance of the adults, but in spite of its important role in nest homeostasis, little is known about whether and how this parameter is regulated by the honeybees. Using miniaturised technology, we measured humidity at different positions inside beehives, and have begun investigating whether this parameter is actively regulated by the workers. We obtained evidence that the honeybee workers influence hive humidity, but do not regulate it precisely. Optimal humidity differs for brood area and nectar stores, resulting in constraints on potential regulation mechanisms in a shared atmosphere. The availability of external water may further impair regulation and the maintenance of optimal humidity levels may be disrupted as a result of trade-offs between temperature regulation and respiratory gas exchanges. Therefore, we argue that workers can only adjust humidity within sub-optimal limits.
Institut für Zoologie, Karl-Franzens-Universität-Graz, Universitätsplatz 2, A-8010 Graz, Austria, E -mail:theniusr©stud.uni-graz.at The behaviour of a honeybee colony depends on many decisions of single honeybees. To understand the behaviour of such a superorganism, the understanding of the individual behaviour is essential. Especially understanding the ways of individual decision making is necessary to explain the ultimate colony behaviour, by which the colony reacts adequately to changes inside and outside of the colony. Freshly emerged honeybees need contact to certain stimuli within the colony (like brood-pheromones) to develop into fully-fledged members of the colony. The first task in a honeybees\' life is the “cleaning” task, which is performed mostly inside the broodnest, preparing cells for the egg-laying queen. Due to this, the presence of young bees in the broodnest area is advantageous for the colony. The self-organised navigation principles of one-day old bees were investigated in a laboratory experiment: We built a comb sized arena with an artificial scalable heat-gradient, with a maximum of about 36°C. We observed the optimum-seeking behaviour and the social interactions of young bees in different shapes of gradients. We found, that bees show different kinds of uphill-going behaviour and different clustering behaviour in dependence of the steepness of the temperature gradient they were exposed to.
University of Plovdiv “Paisii Hilendarski”, Faculty of Biology, Department of Developmental Biology, Section of Genetics, Plovdiv 4000, Bulgaria, E-mail of the corresponding author: geneiv©pu.acad.bg During the last years the electrophoretic investigations of a number of proteins and enzymes play a significant role in the solution of population-genetic and ontogenetic problems but very few electrophoretic analyses have been published yet to describe the age specificity of protein expression in the fat body of the male and female individuals of A. mellifera during their ontogenesis. The aim of this investigation was to elucidate and analyze some peculiarities in the age specificity of the protein expression in the fat body of A. mellifera during the development of the workers and the drones. Using 7.5% PAGE, 184 individual samples of fat body extract were investigated. The workers were studied after laying the eggs in their larval stage (5th day), pre pupal (10th and 11th days) and pupal (14th to 20th days) stages and before flying off the imago (21st day). The drones were studied after laying the eggs in their larval stage (5th and 10th days), pre pupal (14th days) and pupal (18th to 22nd days) stages and before flying off the imago (24th day). A total of 18 protein fractions (SP-1 to SP-18) were expressed in the fat body of male and female individuals during their development. There were found specific proteins for different ages of individuals. Some sex-differences in studied fat body proteins were noticed. The age and stage specific dynamic in the expression of the established proteins of the fat body spectrum was analyzed and commented. Acknowledgements: The study was supported by NFSR of the Ministry of Education and Science of Bulgaria (NoVU AN 2 - 2005) and by “P. Hilendarski” University fund “Scientific research” (No B 20). RNA interference and olfactory memory in the honeybee Lucile Garreau1, Sabrina Oliveira1, Claire Marty 2, Didier Fournier2, Martin Giurfa1, Valérie Raymond-Delpech1, Monique Gauthier1 1 Centre de Recherches sur la Cognition Animale, Université Paul sabatier, 118 route de Narbonne, 31062 Toulouse Cedex 2 Institut de Pharmacologie et de Biologie Structurale, 205 route de Narbonne, 31077 Toulouse cedex E-mail: gauthiem©cict.fr Acetylcholine is involved in olfactory learning in the honeybee through the activation of the nicotinic acetylcholine receptors (nAChRs). Classically, the nAChRs are formed of an assembly of different α and β subunits but in insects, we do not know the molecular composition and the pharmacological properties of these receptors. Nine α and 2 β subunits have been identified recently from the honeybee genome, each of these subunits could potentially combine to the others to form functional nAChRs. For a better understanding of the role of the nAChRs in insect memory, we induced silencing of the α3 subunit using the technique of RNA interference (RNAi). RNAi was injected into the honeybee brain through the median ocellus and the protein expression was evaluated at different times after injection using Western blot analysis. A significant decrease of the α3 protein was observed 6 hours after injection. The effect of the protein suppression was evaluated on the olfactory conditioning of the proboscis extension reflex, using 3 trial olfactory learning. RNAi injection was performed 6 h before the learning session or before the memory test. Saline and RNAi against GFP were used as control. Pre-training injection had no effect on learning performance and induced a response generalization to unknown odorant 24 h after training. RNAi injected 6 h before the retrieval test unexpectedly increased the memory performance. A discriminative olfactory learning is currently under investigation to test generalization phenomenon in honeybees lacking the α3 subunit.
Institut für Zoologie, Karl Franzens Universität Graz, Universitätsplatz 2, A-8010 Graz, Austria, E-mail: karl.crailsheim©uni-graz.at Feeding of protein containing food to honeybee colonies is occasionally recommended in spring to compensate for a lack of pollen. These feeding should supply the nurses with additional protein and thus increase breeding activity after the end of winter. We applied a mixture of yeast and icing sugar containing non toxic stains in different areas of observation hives and analysed the pattern of consumption by behavioural observations and photospectrometic analysis. We found that this food offered was distributed throughout the colony either by direct consumption or by a trophallactic pathway. The stains were found in larval food for various ages as well as in honey. Thus the protein in the artificial food did not go just to the nurses but was fed in no processed form to the larvae and to other bees. The fact that it could be found in honey also indicates a contamination of this bee product with protein. The meaningfulness of protein supplementation for honeybees is discussed controversy in literature. We did not test the effect of the provided protein but could show that the protein is rather uniformly distributed than selectively utilized by a special cohort of bees.
Centre de Recherches sur la Cognition Animale, Université Paul Sabatier, 118 route de Narbonne, 31062 Toulouse cedex, E-mail: gauthiem©cict.fr Individual restrained honeybees were submitted to side-specific tactile learning of the proboscis extension response. In this task, a tactile stimulus delivered to one antenna is associated to a reward of sucrose solution delivered to the proboscis. Two tactile stimuli, A and B, were used as conditioned stimuli in a 6 trial learning paradigm. In the first learning situation, one antenna was stimulated with A and the honeybee was reinforced with sucrose and the other antenna was not stimulated (A+/0). In the second situation, A presented to one side was reinforced and B was presented to the other side non-reinforced (A+/B-). In the third situation, A was learned on one side and B was learned on the other side (A+/B+). During retrieval tests, both stimuli were randomly presented to each antenna 3 h and 24 h after training. Fifteen min. before learning, the animals received a thoracic application (1 μl) of fipronil (0.1 ng) or of acetone solvent (control group). In (A+/0) and (A+/B-) training situations, control animals responded to A on both sides, showing a transfer of information between sides. No generalization of the conditioned response to B- was observed. When honeybees specifically learned two different stimuli on each side (A+/B+), they showed side-specific response patterns, excluding transfer and generalization phenomena. Fipronil-treated honeybees had lower performance than control bees in the (A+/0) and (A+/B+) trainings and did not discriminate between A+ and B- in the (A+/B-) training. In the retrieval tests, these honeybees responded to both stimuli on both sides with a memory loss at 24 h. We conclude that a subletal dose of 0.1 ng/bee deteriorates side-specific learning abilities of honeybees
Institute of Zoology, Martin-Luther-University Halle-Wittenberg, 06099 Halle, Germany. E-mail: holger.scharpenberg©zoologie.uni-halle.de Under queenless conditions worker bees are able to activate their ovaries (Ruttner and Hesse 1981), but only few start oviposition and secretion of a queen-like pheromonal bouquet in their mandibular and Dufour’s glands after a short period of extreme intracolonial selection. Such ‘pseudoqueens’ (Velthuis et al. 1990) can suppress ovary activation and the production of queen-like pheromone signal in other workers (Moritz et al. 2000). In this set of experiments the effect of group size on dominance patterns in queenless groups was tested in petri dishes using freshly emerged and individually labelled bees ranging from 2 to 10 workers. Due to the queenless conditions workers were initially unexposed to 9-ODA and could gradually increase their level of pheromonal production over the experimental time. Recently Moritz et al. (2004) analyzed the dynamics of pheromone production in paired workers. They found that queenless workers engage in a pheromonal contest and compete for reproductive dominance, finally resulting in a stable hierarchy of dominant and subordinate workers. With increasing group size, it is difficult to predict whether workers participate in clear-cut reproductive hierarchies dominated by a single worker or whether they will exhibit a continuous range of pheromonal levels with several dominant workers. The aim of the experiment was to quantify social hierarchies in different group sizes, using the highly sensitive GC-analysis of the mandibular gland pheromones (queen-worker substance ratio). Dependence of the morphological characteristics of the bee venom gland of the honey bee on the parameters of the third tergit Peter Nentchev , Lina Jordanova Trakia University, Stara Zagora, Bulgaria E-mail: Nentchev©hotmail.com ; lina©uni-sz.bg This investigation is one of a set of studies carried out on the morphological characteristics of the bee venom gland of the honey bees in Bulgaria. The aim of the investigation is the establishment of the correlative dependence of the morphological characteristics of the bee venom gland on the parameters of the third tergit of Apis mellifera Sp.L. honey bee in Bulgaria. The investigation has been carried out at the experimental base of Apiculture at the Agricultural Faculty at the Trakia University in Stara Zagora. 87 marked honey bees from a honey bee colony at the age of 30-40 days have been used. The sting, together with the reservoir and the bee venom gland of each mortified bee has been measured. The length of the trunk of the bee venom gland, the length and the width of the reservoir have been measured by a microscope. The volume is calculated by the following formula: V=(0,5.a)².π.b/3, where π=3,14; b and a are the length and the width of the reservoir. The third tergit has been separated and measured following the method of Alpatov. In this investigation the length, the width and the volume of the reservoir have been studied together with the length, the width and the distance between the tentacles as included in this investigation parameters of the third tergit. The statistical calculation of the data includes: the receiving of the basic statistical numbers of all mentioned measurements, the calculation of the coefficients of the correlation between them, the receiving of the regressive equations for all measurements depending on the parameters of the third tergit. Two models for calculation of the regressive equations have been used in the investigation: Yј=µ+Σaіxi, i=1,2,3 (1) Yј=Σaіxi, i=1,2,3 (2) where µ is the average number for the studied population, Yј is one of the measurements of the bee venom gland, the variable quantities xi are the width, the length and the distance between the tentacles as parameters of the third tergit, together with coefficients in the equation ai. Statistica for Windows has been used for the calculations. The established coefficients of the correlation between the used measurements and the linear regressive equations are statistically reliable. The established correlations and equations could be used for the selection of honey bee colonies by the signs of the honey bee venom gland. They could also be used for making a forecast of the measurements of a honey bee colony and for a forecast of the yield of honey bee venom. A technique for measuring temperature distributions in the comb of honeybees (Apis mellifera) Matthias Becher, Robin FA Moritz Institute of Zoology, Martin-Luther University Halle-Wittenberg, Hoher Weg 4, 06099 Halle (Saale), Germany E-mail: becher©zoologie.uni-halle.de In order to save energy, honey bees (Apis mellifera) are able to form clusters, when ambient temperatures fall below a threshold of about 18°C. Those winter clusters consist of an outer mantle insulating a warmer core region and are maintained by the consumption of honey stores for metabolic heat production. To analyse temporal and spatial temperature distribution in honey bee combs, we constructed a precise thermo-device, providing us constantly with temperature data of 768 cells. These data are coupled with a simultaneous video record of the comb allowing for the combination of worker allocation on and temperature distribution in the comb. Here we test different group sizes of clustering bees on the heat transfer to the comb under cool temperature conditions. Efficient heat accumulation requires a least number of individuals. We investigated the relationship between number and distribution of the bees and steepness of the temperature gradient on the test-comb in dependence of differing ambient temperatures. Candidate genes involved in the honeybee social behaviors Hideaki Takeuchi Department of Biological Sciences, Graduate School of Science, The University of Tokyo, Bunkyo-ku, Tokyo 113-0033, Japan; E-mail: takeuchi©biol.s.u-tokyo.ac.jp The honeybee is a social insect and its colony consists of a queen, workers and drones that have different roles. In addition, various complex social behaviors, such as communications and divisions of labor, are performed by colony members to maintain colony activities. Mushroom body (MB) is one of insect brain structures, which is important for learning, memory and sensory integration. Honeybee MB function is believed to be closely associated with honeybee social behaviors for the following reasons: 1) The MBs of the aculeate Hymenoptera, including the honeybee, are more prominent compared with those of other insects. 2) Honeybee MBs have a high degree of structural plasticity and the volume of the neuropil varies according to the division of labor. To clarify molecular basis underlying honeybee MBs function, we identified over 20 genes expressed selectively in the MBs of the honeybee brain using combination of differential display and cDNA microarray method. In this talk, I will present the expression pattern of these MB-selective genes and discuss possible function of these genes in comparison with those of their orthologues in non-social insects/invertabrate. We also study about visual learning and chromatic adaptation in the harnessed honeybee and propose our strategy to examine involvement of these genes in the honeybee visual behaviors. Reference Hori S, Takeuchi H, et al. (2006) Associative visual learning, color discrimination, and chromatic adaptation in the harnessed honeybee. Journal of Comparative Physiology A., in press.
AFSSA, E-mail: m.aubert©afssa.fr Field observations on the incidence of honey bee virus infections suggest that honey bee individuals as well as colonies may suffer severely from the infections. Furthermore, it seems likely from field reports that the development of overt infections is context dependent. In other words, honey bees may carry infections but suffer little or no consequences, but given other conditions the infection may become overt and the impact fatal at colony level, even from rather limited effects at the individual bee level. Nevertheless, it is necessary to unravel the causal relationship between colony deaths and virus infections, since available field data are not conclusive. For many bee viruses, pathogenicity is still poorly understood. However, field observations show that several virus infections are associated with honey bees symptoms and significant mortality. Viruses are among the simplest parasitic forms, they are widely spread and always ready to adapt themselves to new conditions – included those created by beekeepers. Moreover, the recent introduction of Varroa destructor demonstrated that conditions and routes of transmission may be dramatically changed. Problems with honey bee viruses are real, they require more appropriate studies to be assessed and controlled. Colony mortality and bee viruses Hélène Berthoud, Anton Imdorf, Monika Haueter and Jean-Daniel Charrière Swiss Bee Research Centre, Agroscope Liebefeld-Posieux, Schwarzenburgstrasse 161, 3003 Bern, Switzerland E-mail: helene.berthoud©alp.admin.ch Sensitive molecular detection methods showed the presence of bee viruses in apparently healthy colonies, sometimes even with high virus titres (Tentcheva et al. 2004). The aim of our study was to compare prevalence of viruses in healthy and diseased colonies. At the end of the winter, beekeepers announced apiaries with abnormally high colony mortality. Bee samples were taken from dead, weak or apparently healthy colonies in these apiaries and from healthy colonies in control apiaries. Pools of 50 bees were analysed for the presence of Acute Bee Paralysis Virus (ABPV), Deformed Wing Virus (DWV), Chronic Bee Praralysis Virus (CBPV) and Kashmir Bee Virus (KBV). Positive samples were then analysed with a quantitative RT-PCR method. Results showed that neither CBPV nor KBV were detected. In control apiaries, ABPV was never found and low DWV titers were found in only 25% of the colonies tested. In diseased apiaries, all colonies were DWV positive with significantly higher virus loads. Moreover, about 70% of colonies were also ABPV positive. The differences observed between healthy and diseased colonies indicate that ABPV and DWV could play a role in colony mortality during the winter. A reduced bee life span known to be associated with virus infections could explain the colony mortality. However, the factors inducing high virus titres, like mite infestation, are to be further studied.
1Bee institute Kirchhain, Erlenstraße 9, D-35274 Kirchhain, garridoc©llh.hessen.de 2 State Institute of Apiculture Hohen Neuendorf, Friedrich-Engels-Straße 32, 16540 Hohen Neuendorf Within the scope of the German varroosis tolerance breeding project we compared five bee lines concerning the virus infections. The colonies were kept during two years without treatment against varroosis. Bee samples were taken from the colonies throughout this period. We analyzed four viruses (ABPV, KBV, SBV and DWV) by means of PCR technique. Additionally, the viral load of ABPV and DWV was analyzed by means of RT-PCR technique. We found differences in the patterns of virus infections of the different bee lines. The correlation to colony development, overwintering performances and Varroa infestation level is discussed.
1Institut für Virologie, Fachbereich Veterinärmedizin, Justus Liebig Universität Giessen, Frankfurter Str. 107, D-35392 Giessen, Germany 2Landesbetrieb Landwirtschaft Hessen, Bieneninstitut Kirchhain, Erlenstr. 9, D-35274 Kirchhain, Germany
The acute bee paralysis virus (ABPV) is pathogenic for honey bees (Apis mellifera). ABPV is a small, single-stranded RNA virus with positive polarity. Recently ABPV was classified as a tentative species of the genus Cripavirus (Fam. Dicistroviridae). It was suggested that the virus is firmly associated with the bee-parasitic mite Varroa destructor. High amounts of ABPV were found in bees from colonies which died of varroosis. The parasite is believed to represent a vector for ABPV and to be important for fatal ABPV outbreaks. Here we report a field survey of 11 German apiaries which were followed up for two years. In autumn 2004 and 2005 worker bees were skimmed off from combs of ten bee colonies per apiary. In march the survivorship from the previous winter of each colony was recorded. The mite infestation in autumn and the colony strength before and after winter was registered. Out of the group of the surviving hives three colonies per apiary were randomly selected for analysis of their ABPV-load. All bee samples which derived from collapsed colonies and heavily damaged bee yards were assessed for ABPV. For this purpose a SYBR-green real-time PCR assay was developed. Much variation of the ABPV burden occurred during the two years between the colonies and between the yards. The ABPV burden was linked to the number of mites. The presumption of a linkage between the ABPV load and the colony strength after winter was investigated. Interestingly, higher ABPV loads were followed by colony deaths. Our data suggest that the amount of ABPV in autumn can be correlated with poor outcome in winter. Prevalence and phylogeny of Kakugo virus identified from aggressive worker honeybees (Apis mellifera L.) under various colony conditions Tomoko Fujiyuki1, Seii Ohka2, Hideaki Takeuchi1, Masato Ono3, Akio Nomoto2, Takeo Kubo1 1 Graduate School of Science and 2Graduate School of Medicine, The University of Tokyo, Japan. 3Honeybee Science Research Center, Tamagawa University, Japan. E-mail: fujiyuki©biol.s.u-tokyo.ac.jp We previously identified a novel insect picorna-like virus, termed Kakugo virus (KV), from the brains of aggressive worker honeybees that had counterattacked a giant hornet. To examine the relation between KV infection and aggressive worker behavior, we surveyed the prevalence of KV in various worker populations by quantifying KV genomic RNA. KV was detected specifically from aggressive workers in some colonies, while it was detected also from other worker populations in other colonies where the amount of KV detected in the workers was relatively high. This finding suggests that in the primary infection phase KV infection is attacker specific, whereas in the late phase KV is infectious to various worker populations. To investigate whether KV strains detected were identical, phylogenetic analysis was performed. There was a less than 2% difference in the RNA-dependent RNA polymerase (RdRp) sequences between KV strains from aggressive workers and those from other worker populations, and these strains had approximately 6% and 15% sequence differences in the RdRp region from deformed wing virus and Varroa destructor virus-1, respectively. These results suggest that all of the viruses we detected were virtually the same KV and KV represents a closely related but distinct viral strain from the other two viruses. We also found some of the KV-infected colonies were infested with Varroa mites and sequences of the KV strains detected from the mites were the same as those detected from the workers of the same colonies, suggesting that the mites mediate KV prevalence in the honeybee colonies. The pitfalls of diagnosis interpretation in honey bee pathology. The case of deformed wing virus (DWV) Gauthier Laurent Montpellier University, E-mail: gauthier©univ-montp2.fr Among the fifteen distinct viruses identified in Apis mellifera L., the deformed wing virus (DWV) is one of the most prevalent in honey bee colonies. The high prevalence of DWV is likely correlated to its ability to be transmitted by the mite Varroa destructor. The PCR amplification of DWV negative RNA strands in mites and the tremendous amounts of DWV genome copies recorded from mites argue for the replication of DWV in varroa and in bee as well. Besides, there is strong evidence that DWV is also transmitted between individuals of the colony, either horizontally by food exchange or vertically through the queen’s eggs. We attempted to measured DWV RNA loads in 360 seemingly healthy honey bee colonies from pools of 100 bees using quantitative PCR. Our data showed that honey bee colonies can tolerate very high loads of viruses without displaying external clinical signs. We further identified DWV RNA in several bee organs using the in situ hybridization technique and showed that queen and drone fertility could be impaired by such infection. In queen, the fat body cells were particularly infected while in drone, the whole reproductive tract was clearly stained with the DWV probe. Moreover, in crippled winged individuals from where very high DWV RNA genome copies were recorded, the digestive tract was heavily stained with the DWV probe, indicating a probable negative effect of DWV infection on the digestive function of the bee. Our data strongly support that DWV produces pathogenic effects in severely infected individuals from the colony. However, when considering the colony level, these deleterious effects observed on individual bees might not always have an impact on the colony fitness.
AFSSA France, E-mail: m.ribiere©afssa.fr Knowledge of the spreading mechanism of honey bee pathogens in the hive is crucial to our understanding of the dynamics of bee disease. The aim of our study was to assess the presence of infectious CBPV in bee excreta and to evaluate its possible role as an indirect route of infection. Batches of or individual infected bees were produced by experimental inoculation with purified virus or collected from colonies with paralysed bees. CBPV in bees (bee heads) or faeces (fresh or absorbed onto paper) was detected by reverse transcription-polymerase chain reaction (RT-PCR). CBPV RNA was detected in faeces of naturally and experimentally infected bees and on the sheets of paper that were used for covering the floor of units that had contained artificially CBPV infected bees or for covering the floor of naturally infected colony. Additionally CBPV infectivity was assessed by intra-thoracic inoculation of naive bees with faeces extracts or by placing naive bees in cages previously occupied by contaminated individuals. Both procedures entailed overt disease in naive bees. To our knowledge this is the first experimental confirmation that infectious CBPV particles excreted in faeces of infected bees can infect naive bees by mere contact and provoke overt disease.
AFSSA, E-mail: m.aubert©afssa.fr Built on EurBee initiatives, the European BRAVE project (Bee Research And Virology in Europe ) (duration : the year 2005) was aimed at knowledge transfer between experts in insect virology and scientists involved in research on bees and related species. Furthermore, the project aim was to synthesise the current knowledge required for protecting the honey bee and related pollinator insects from virus diseases and to propose a framework for future research programmes in this field. More than 60 world experts exchanged during a meeting in Sophia-Antipolis (France) in April 2005. They explored the taxonomy of bee viruses, diagnostic techniques and their appropriateness. Genetics, physiology and behaviour of honey bees were studied in relation to their resistance to virus infections. The association of virus infections with parasites such as Varroa destructor or Nosema apis, and the possible depression of the honey bee immune response by exposure to sub-lethal doses of pesticides were also raised. The evolutionary epidemiology of virus diseases, current information on the incidence, distribution and impact of honey bee viruses, the management of bee diseases including the regulatory mechanisms governing the world exchanges were all covered. The proceedings of this meeting have been published ww.entom.slu.se/BRAVE/publications.htm). They include the scientific texts and the recommendations elaborated by the working groups and approved in plenary sessions. A workshop meeting took place later at which several experts have prepared a synthesis of current knowledge in these areas. This task will be published in the form of a book entitled "Virology and the Honey Bee" whose publication is expected at the end of this year. The European BRAVE project will have produced tangible results : the proceedings of the BRAVE plenary scientific meeting and the comprehensive book "Virology and the Honey Bee".
Warsaw Agricultural University, Faculty of Veterinary Medicine, Department of Clinical Sciences, E-mail: grazyna_topolska©sggw.pl Black queen cell virus (BQCV) has been found to be a cause of death of queen prepupae and pupae. In adult bees it almost invariably multiplies only in those individuals that are also infected with Nosema apis. Recent studies conducted in Maryland (USA) revealed the presence of the virus in 100% of queen bees which were collected for examination from randomly chosen colonies. From our previous investigations of queen cells, we draw the conclusion that queen rearing apiaries can differ considerably as to the level of BQCV infection of reared queen bees. In this work we examined for presence of BQCV unmated queens sold to bee keepers by four queen rearing apiaries. The queens were reared in May and August. BQCV was found only in the queens reared in one apiary but both in May and August. These queens were free from Nosema apis spores so we concluded that they had become infected with the virus through ingestion of contaminated food during the larval rather than the mature stage. STUDY OF CHRONIC BEE PARALYSIS VIRUS (CBPV) REPARTITION IN HONEY BEE (APIS MELLIFERA) COLONIES Magali Ribière*, Philippe Blanchard*, Frank Schurr, Perrine Lallemand, Olivier Celle, Violaine Olivier, Jean-Paul Faucon. AFSSA France, m.ribiere©afssa.fr, p.blanchard©afssa.fr Knowledge of the virus distribution in bee colonies and of the kinetics of virus load are crucial to our understanding of Chronic paralysis infection dynamic. This study investigated CBPV infection in several bee colonies either symptomatic or asymptomatic of the same apiary. The CBPV genomic loads of a variety of brood samples (with eggs, larvae or pupae), home, guard, forager bees, and symptomatic and dead bees collected from the flight board were quantified by a real-time TaqMan RT-PCR assay. Results obtained from symptomatic or dead bees from colonies with Chronic paralysis confirm the correlation between high CBPV genomic load and pathology expression. Surprisingly, highest CBPV genomic loads were not observed in the group of the oldest bees (the foragers) but in guard bees, which address the question of their role in the infection. Thus, similar investigations of CBPV infection are now being conducted on other apiaries in an attempt to draw out the risk factors associated with CBPV expression. Evolution of virus distribution in the colony and determination of CBPV genomic load level in correlation with CBPV expression are discussed
AFSSA France, E-mail: p.blanchard©afssa.fr A two step real-time RT-PCR assay, based on TaqMan technology using a fluorescent probe (FAM-TAMRA) was developed to quantify chronic bee paralysis virus (CBPV) genome in bee samples. According to an absolute standard curve obtained with a plasmid containing a partial sequence of CBPV, this assay provided linear detection over a 7-log range (R2>0.99) with a sensitivity of 100 copies, confirmed by reliable inter-assay and intra-assay reproducibility. Standardisation including crushing, RNA purification and cDNAs synthesis was also validated. In order to evaluate the CBPV TaqMan methodology, the CBPV genomic load was quantified in bee samples coming from an experimental infection by contact. Up to 1.9 x 1010 CBPV copies were revealed in a segment of insect body (head, thorax and abdomen) while lower CBPV genomic load was observed in dissected organs as mandibular and hypopharyngeal glands, brain and alimentary canal (up to 7.2 x 106 CBPV copies). These preliminary results validate this method for chronic bee paralysis virus quantification.
AFSSA France, E-mail: v.olivier©afssa.fr Most viruses of the honey bee are classified as “picorna-like virus”, in the Cripavirus genus (Dicistroviridae family) or in the Iflavirus genus. Chronic bee paralysis virus (CBPV) is a single-stranded RNA virus as “picorna-like viruses” are. However, CBPV has a multipartite RNA genome with two main RNA and differs especially from these viruses at genomic and phylogenetic levels. About 81% of the genomic sequence of the two main RNA have already been determined by different techniques such as cDNA library and 5’RACE. Sequencing is complexified by the apparent blockage of the 3’ end of RNA as some other RNA virus genomes are (Nodavirus, Tobamovirus or Bromovirus). Our first phylogenetic analysis, carried on nucleic sequence of the putative RNA dependant RNA polymerase, showed no significant similarity between CBPV and other honey bee viruses. However, few similarities between CBPV and viruses belonging to the Nodaviridae taxon (insect and fish viruses) have been shown. The complete characterisation of the CBPV genus by sequencing and phylogenetic analysis is in progress. Transmission routes of Deformed wing virus Constanze Yue and Elke Genersch Institute for Bee Research, Friedrich-Engels-Str. 32, 16540 Hohen Neuendorf, Germany E-mail: elke.genersch©rz.hu-berlin.de Deformed wing virus (DWV) is a plus-stranded RNA-virus belonging to the floating genus Iflavirus. It is pathogenic for honeybees and could also be demonstrated in bumble bees recently. In most cases, DWV-infected bees do not exhibit overt signs of disease. In colonies infested by the ectoparasitic mite Varroa destructor DWV-infections can be associated with typical clinical symptoms like crippled wings, shortened and bloated abdomen, and discoloration. The recent publication of the genomic sequence of DWV was followed by the development of several RT-PCR protocols for the highly sensitive and specific detection of this virus. Subsequently, numerous studies on the incidence and prevalence of DWV in honeybees and honeybee colonies were published. Still, little is known so far about the molecular pathogenesis of DWV. We investigated the first step in the pathogenic process, the transmission of the virus. We found evidence for vertical transmission of DWV through drones, horizontal virus transmission to larvae through feeding, and vectorial transmission to larvae and adult bees with Varroa destructor acting as possible virus vector. Interestingly, the occurrence of crippled bees correlated with the virus’ ability to replicate in mites. This phenomenon needs further investigation. Prevalence of six honeybee viruses in beehives collected at different Austrian locations during different seasons, and correlation with non-viral diseases see fulltext Hemma Köglberger1, Irmgard Derakhshifar1, Jolanta Kolodziejek2, Hans Homola², and Norbert Nowotny2 Institute for Apiculture, Austrian Agency for Health and Food Safety, A-1226 Vienna, Austria1 Zoonoses and Emerging Infections Group, Clinical Virology, Clinical Department of Diagnostic Imaging, Infectious Diseases and Clinical Pathology, University of Veterinary Medicine, Vienna, A-1210 Vienna, Austria²
KBV was never detected, and CBPV just in one case in winter (2%), while RNA of all other honeybee viruses investigated were identified in varying percentages: ABPV: 2% (May), 57% (July), 52% (September), and 39% (winter). SBV: 45% (May), 72% (July), 50% (September), and 20% (winter). BQCV: 66% (May), 46% (July), 33% (September), and 14% (winter). DWV: 26% (May), 11% (July), 24% (September), and 43% (winter). The first results of diagnostics of viral diseases of bees in Slovakia T. Čermaková, J.Habovstiaková , M.Kantiková Slovak centre of agricultural research Nitra, State veterinary and food institute Dolny Kubin Deformed wing virus (DWV) is the most commonly observed and best know honey bee virus in Europe. This virus was initially isolated from adult honeybees (Apis melifera) from Japan. DWV is 30 nm isometric particles containg a single-stranded positive RNA, which is assigned to the genus Iflavirus. In recent study using molecular techniques, was DWV detected in all life stages (eggs, larvae, pupae, adults) of honey bees and frequency of DWV-infected colonies was increased from spring to autumn. DWV can cause morfological deformities, as are: wing deformity, malformed appendages, shortened abdomens and miscolouring. DWV generally persists as a latent infection with no apparent symptoms. In adult bees, during the year DWV was found at least once in 97% of the apiaries and for pupae it was 94% of the apiaries. The question is, what is reason of change from a latent infection to productive infection, which results in collaps of all collonies. The disease and mortality caused by DWV have been associated with severe infestation of the ectoparasitic mite Varroa destructor. This mite is the most commonly observed parasit of bees in the world. Generally, two hypotheses are cited that could explain the deleterious effects of varoa parasitism on honey bee colony decline. First, the mite can act as a vector and can directly inject virus particle into the insect hemolymph. Second, the mite can also trigger virus replication by a simple mechanical effect, cuticle piercing, or by injection of external proteins into the insect hemolymph. The latter hypothesis is supported by several studies that demonstrated that there was reactivation of viruses already present in the insect following experimental inoculation. Several techniques have been employed for detecting bee viruses, including indirect fluorescent-antibody analysis (IFAT), agarose gel immunodiffusion, an enzyme-linked immunosorbent assay (ELISA), Southern hybridization, reverse transcription-PCR (RT-PCR) and real-time quantitave RT-PCR.
Virus Laboratory, The Hebrew University of Jerusalem, Faculty of Agricultural, Food and Environmental Quality Sciences, Rehovot 76100, Israel. E-mail: sela©agri.huji.ac.il , eyalmm©hotmail.com , tanne©agri.huji.ac.il Divergence among individuals of the same species may be linked to positional retrotransposition into different loci in different individuals. Here we add to recent reports indicating that individual variance occurs due to the integration of non-retroviral (potyviral) RNAs into the host genome via RNA recombination followed by retrotransposition. We report that in bees (Apis mellifera), approximately 30% of all tested populations carry a segment of a dicistrovirus in their genome and have thus become virus-resistant. Reciprocally, segments of host sequences have been found within defective-interfering-like sequences of a dicistrovirus. Similarly, host sequences were found fused to potyviral sequences, previously described integrated into their host genome. A potential, continuous RNA exchange leading to divergence is discussed. |