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Apidologie, 2012 - Vol. 43

Association of Institutes for Bee Research

Report of the 59th Seminar in Bonn

27 - 29 March 2012

Arbeitsgemeinschaft der Institute fr Bienenforschung e.V.

59. Jahrestagung in Bonn

vom 27.-29. Mrz 2012

Ecology, pollination, plant protection

kologie, Bestubung, Pflanzenschutz

1. Biology of reproduction in stingless bees: open questions. Fortpflanzungsbiologie stachelloser Bienen: offene Fragen. E. Engels, W. Engels 2. The role of bees as pollinators of bromeliads with ornithophilous flower syndrome. Rolle von Bienen als Bestuber bei Bromelien mit ornithophilem Bltensyndrom. A. Zillikens, J. Steiner 3. Quantification of pollen grains in honey. Festellen der Pollen Menge in Honig. T. Blacquire, C. Hok-A-Hin, C. van Dooremalen 4. Healthy colonies - Does an improved consultation of beekeepers lead to lower colony-losses? Gesunde Bienenvlker- Fhrt eine verbesserte Beratung der Imker zu geringeren Vlkerverlusten? G. Brunnemann- Stubbe, V. Poker, R. Bchler

Physiology, behavior

Physiologie, Verhalten

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5. The serotonin 5-HT2 receptor system of the honeybee (Apis mellifera). Das Serotonin-5-HT2-Rezeptor-System der Honigbiene (Apis mellifera). W. Blenau, M. Thamm, D. Rolke, A. Baumann 6.The loss of bees an important and puzzling parameter in population dynamics of honey bee colonies. Der Bienenabgang - eine wichtige und rtselhafte Gre in der Populationsdynamik des Bienenvolkes. G. Liebig, P. Aumeier, O. Boecking Bees-Pathology

Bienenpathologie

7. Biological control of Varroa destructor using entomopathogenic fungi. Biologische Bekmpfung von Varroa destructor mit entomopathogenen Pilzen. J. Pflugfelder, P. Gallmann 8. Identification of first Toxins of Paenibacillus larvae. Identifizierung der ersten Toxine von Paenibacillus larvae. A. Fnfhaus, L. Poppinga, E. Genersch 9. CBP, a chitin-binding protein of Paenibacillus larvae has an important role in AFB pathogenesis. CBP, ein Chitin-bindendes Protein von Paenibacillus larvae spielt eine wichtige Rolle in der Pathogenese der AFB. E. Garcia-Gonzalez, A. Jakubowska, E. Genersch 10. Functional analysis of the virulence factor SplA, a Paenibacillus larvae ERIC II specific S-layer protein. Funktionelle Analyse des Virulenzfaktors SplA, einem Paenibacillus larvae ERIC II spezifischen S-layer Protein. L. Poppinga, B. Janesch, A. Fnfhaus, G. Sekot, E. Garcia-Gonzalez, G. Hertlein, K. Hedtke, C. Schffer, E. Genersch 11. Alternative antibiotics against nosemosis. Alternative Antibiotika gegen die Nosemose. S. Gisder, E. Genersch

Symposium of the collaborative project Reference system for a vital bee colony: FIT

BEE

12. Effects of chronically administered sublethal thiacloprid does on the vitality of honeybee colonies. Auswirkungen chronischer Verftterung sublethaler Thiacloprismengen auf die Vitalitt von Bienenvlkern. R. Siede, L. Faust, C. Maus, B. Grnewald, R. Bchler 13. Residues of insecticides and fungicides in nectar and honey after using different application technologies in blooming Brassica napus.

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Die Wirkstoffbelastung von Nektar und Honig nach unterschiedlichen Pflanzenschutz-mittelapplikationsverfahren im blhenden Raps. K. Wallner, K. Schmitz

Posters

Ecology, pollination, plant protection

kologie, Bestubung, Pflanzenschutz

14. Foraging and pollination behaviour of Apis mellifera adansonii Latr. (Hymenoptera, Apidae) on Gossypium hirsutum L. (Malvaceae) flowers at Dang (Ngaoundr, Cameroon). Sammel- und Bestubungsverhalten von Apis mellifera adansonii Latr. (Hymenoptera, Apidae) auf Gossypium hirsutum L. (Malvaceae) in Kamerun. M. Sanda, F.-N. Tchuenguem Fohouo, D. Brckner 15. Drift of Dust containing insecticides during sowing of maize- exposure and effects on bees (Apis mellifera L.). Drift von insektizidhaltigen Stuben whrend Maisaussaat- Exposition und Auswirkung auf Bienenvlker (Apis mellifera L.). P.-Th. Georgiadis, J. Pistorius, U. Heimbach

16. Particle size and toxicity of abraided seed coating? Beizstaub - Partikelgren und ihre Giftigkeit fr Honigbienen? H. Mhlhuser, K. Wallner 17. A method for testing effects of honey components on bee development. Eine Methode zur Untersuchung des Einflusses von Honigbestandteilen auf die Entwicklung von Bienen. B. Lichtenberg-Kraag, E. Genersch 18. Characterization of regional honey as a reflection of the occuring flora of an area. Charakterisierung regionaler Honige als Spiegelbild des Pflanzenvorkommens eines Gebietes. E. Etzold, B. Lichtenberg-Kraag

19. A colony based drone keeping method allows analysis of sperm characteristics in honey bee drones. Eine koloniegesttzte Drohnenhaltungsmethode zur Bewertung von Spermien. C. Vogt, N. Hcherl, S. Hrtel 20. Effects of honey bee colonies (Apis mellifera L.) following a granular application of Santana containing the active clothianidin in maize in the years 2010 and 2011. Effekte auf Honigbienenvlker (Apis mellifera L.) bei Anwendung des Bodengranulats Santana mit dem Wirkstoff Clothianidin im Mais in den Jahren 2010 und 2011. J. Werres, D. Wittmann 21. Reproductive Biology and Flower Visitors of the Wild Service Tree (Sorbus torminalis L. CRANTZ). Reproduktionsbiologie und Bltenbesucher der Elsbeere (Sorbus torminalis L. CRANTZ). J. Werres, D. Wittmann

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Physiology, Behaviour

Physiologie, Verhalten

22. Residues in Guttation droplets- potential risk for honey bees (Apis mellifera L.)? Field trials with set up of bee colonies at field edges of seed treated Winter oilseed rape and cereals. Wirkstoffhaltige Guttationstropfen- Auswirkungen auf Honigbienen (Apis mellifera L.)? Feldversuche mit feldrandnaher Aufstellung an gebeiztem Raps und Getreide. I. Joachimsmeier, J. Pistorius, D. Schenke, W. Kirchner 23. Ionic current modulation of honeybee mushroom body and antennal lobe neurons. Modulationen von Ionenstrmen in Pilzkrper und Antennal Lobus Neuronen der Honigbiene. S. Himmelreich, B. Grnewald 24. Apis florea colony surface temperature changes in response to disturbance. nderungen der Oberflchentemperatur bei Strungen von Apis florea Kolonien. N. Haddad , A. Batainha, J. Tautz , S. Fuchs 25. For drones , the serious side of life begins at day six. An Tag sechs beginnt fr Drohnen der Ernst des Lebens. N.Hirtz, D. Ahrens, H. Gtschenberger,N.Hcherl, M. Pahl, J. Tautz 26. Apis mellifera sperm cells shrink during storage. Apis mellifera-Spermien schrumpfen whrend der Lagerung. J. Wegener, K. Bienefeld 27. Influence of social cues on decision making of honeybees in a temperature gradient. Der Einfluss eines sozialen Gradienten auf die Entscheidung von Bienen in einem Temperaturgradienten. S. Hahshold, M. Szopek, G. Radspieler, R. Thenius, T. Schmickl, K. Crailsheim 28. Do artificially reared bees (Apis mellifera carnica) have the ability to nurse? Haben knstlich aufgezogene Bienen (Apis mellifera carnica) die Fhigkeit Brut zu pflegen? K. Tschigg, U. Riessberger-Gall, K. Crailsheim 29. Stability of behavioural classes in young honeybees. Stabilitt von Verhaltenstypen bei jungen Honigbienen. G. Radspieler, R. Thenius, T. Schmickl, K. Crailsheim Bees- Pathology

Bienenpathologie

30. Residues in Guttation droplets - potential risk for honey bees (Apis mellifera L.)? A worst case semi-field scenario in maize with special consideration of impact on bee brood and brood development. Wirkstoffhaltige Guttationstropfen Auswirkungen auf Honigbienen (Apis mellifera L.)? Halbfreilandversuche in Mais mit besonderer Betrachtung der Brutentwicklung.

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M. Frommberger, J. Pistorius, A. Schier, I. Joachimsmeier, D. Schenke 31. The long road of hyperthermia in control of varroa. Der lange Weg der Hyperthermie bei der Varroa-Kontrolle. W. Engels 32. Preliminary results of honeybee winter losses in Austria in 2010-2012. Zwischenergebnisse der Untersuchungen von Winterverlusten in sterreich 2010-2012. H.Kglberger, I. Derakhshifar, R. Moosbeckhofer 33. Effects of pollen diets on the longevity and development of hypopharyngeal glands of honeybees. Einfluss einseitiger Pollenernhrung auf die Lebensdauer und Entwicklung der Hypopharynxdrse der Honigbiene. E.Blum, I.Illies, J.Tautz 34.Analysis of oxalic acid distribution in colonies of Apis mellifera by computed Tomography. Untersuchungen zur Verteilung von Oxalsure im Bienenvolk von Apis mellifera durch Computertomographie. E. Rademacher, A. Fahlberg, M. Harz 35. Short and sweet - optimal performance of formic acid-based treatment in honey bee colonies. Kurz und bndig optimale Anwendung von Ameisensure im Bienenvolk. P. Beinert, P. Aumeier, G. Liebig, C. Lffler, H. Aupperle, W. H. Kirchner 36. Systems biology potential application in the system Paenibacillus larvae. Systembiologie Anwendungsmglichkeiten im System Paenibacillus larvae. A. Fnfhaus, E. Genersch 37. In vitro-gene expression of Nosema spp. In vitro-Genexpression von Nosema spp. S. Gisder, E. Genersch 38. Potential siderophore in Paenibacillus larvae. Potentielles Siderophore in Paenibacillus larvae. G. Hertlein, E. Garcia-Gonzalez, L. Poppinga, A. Fnfhaus, E. Genersch 39. Successful expression of heterologous proteins in Paenibacillus larvae. Erfolgreiche Expression heterologer Proteine in Paenibacillus larvae. L. Poppinga, E. Genersch 40. Functional characterization of secondary metabolites of Paenibacillus larvae. Funktionelle Charakterisierung von Sekundr-Metaboliten von Paenibacillus larvae. E. Garcia-Gonzalez, S. Mller, S. Gisder, R. Borriss, R. Sssmuth, E. Genersch 41. Cell culture models for DWV. Zellkulturmodelle fr DWV. N. Mckel, E. Genersch 42. Efficacy of the varroa treatment with half of the recommended doses of rotenone in stripes in Rijeka, Croatia. Wirkung der Varroabehandlung mit der Hlfte der empfohlenen Dosis Rotenon in Rijeka, Kroatien. M. Katelan, D. ekulja

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43. Comparison of the efficacy of varroa treatment with rotenone in powder solution prepared by dilution of the powdered product Cube Powder Fish Toxicant, TIFA International LLC, with efficacy of varroa treatment with Check Mite+ and originally packed rotenone in stripes in Rijeka, Croatia. Das Varroa-Behandlungsmittel Rotenon: Vergleich zwischen der Wirkung einer Pulver-Lsung (Cube Powder Fish Toxicant, TIFA International LLC), Check Mite+ und den original verpackten Rotenonstreifen in Rijeka, Kroatien. D. ekulja, S. Petrovi, M. Vukovi, P. Modruan 44. Efficacy of a single Varroa treatment with preparative Bee Vital Hive Clean at the end of summer 2010 in Rijeka, Croatia. Wirkung einer einmaligen Varroabehandlung mit Bee Vital Hive Clean Ende Sommer 2010 in Rijeka, Kroatien. V. Tiak, D. ekulja Other themes

Andere Themen

45. A proposal of methods to evaluate the hazards of hive coats for honey bees. Ein Methodenvorschlag zur Prfung von Beutenansichten auf Bienengefhrlichkeit. I.Illies, K.Wallner 46. Bee legs and feathers inspires textil engineering. Bienenbeine und Federn inspirieren textile Ingenieurwissenschaft. V. Grabarek, H. Schmied, D. Wittmann 47. Attachment devices of some male Coelioxys species. Haltestrukturen bei Mnnchen einiger Kegelbienen-Arten. M.Piefke, D.Wittmann 48. Geometrical and physical characteristics of oil bee scopa (Epicharis dejeanii, Lepeletier, 1841) a bionic approach. Geometrische und physikalische Eigenschaften der Scopa einer lbiene (Epicharis dejeanii, Lepeletier, 1841) - Ein bionischer Ansatz. L. Pastrik, U.Wilhelm, D. Wittmann

Abstracts

Talks

1. Biology of reproduction in stingless bees: open questions. E. Engels1,2, W. Engels1 (1Universitt Tbingen, Institut fr Evolution und kologie, Auf der Morgenstelle 28, 72076, Tbingen, Germany; 2 Universidade de Sao Paulo)

In neotropical Trigonine stingless bees Sakagami et al. discovered that workers oviposit regularly in queenright colonies. They produced mostly large yolk-rich alimentary or trophic and few slender eggs. The big ones are deposited at the rim of a nearly finished brood cell and eaten by the queen at the beginning of her ovipositioning cycle. The slender

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haploid eggs are smuggled into brood cells already containing a queenlaid egg and develop into males. In Scaptotrigona postica we found that nurse-aged workers 12 to 20 days old are capable of producing one or two large alimentary eggs. The two modes of oogenesis and positioning behavior of the workers are supposedly guided by yet unknown hormonal conditions. - Queens are produced nearly all over the year and reared in large brood cells at the margin of brood combs. Newly emerged virgins hid in corners of the involucrum. They are attacked by workers and usually are executed. Only a few have a chance to survive, in supersedure of the old dominant queen or in joining a swarm to a nearby filial nest. At an age of 12 to 15 days after only these virgins will undertake a short nuptial light from their home nest. In their mother colony they first have to perform during several days an inthronisation dance. After their acceptance the old queen is killed by the workers. In filial nests often several virgins have to fight until one remains. How the princess virgins are selected by the worker community is completely unknown. - In queenright colonies the queen mother enters the brood nest only for short oviposition cycles. She takes a bit of the deposited provisions in brood cells under final construction and also eats up all present alimentary eggs. She is repeatedly encircled by nurse workers and then vibrates her wings, probably to distribute a yet unidentified dominating scent from her tergal glands. In fact a lot of chemo-communication in stingless bee reproduction is demanding research.

2. The role of bees as pollinators of bromeliads with ornithophilous flower

syndrome. A. Zillikens1, J. Steiner2 (1 Universitt Tbingen, Medizinisch Naturwissenschaftliches Forschungszentrum, Ob dem Himmelreich 7, 72074 Tbingen, Germany; 2 Universidade Federal de Santa Catarina, Dept. BEG, Campus Trindade, 88.040-900 Florianpolis, SC, Brazil)

In Atlantic forests at Santa Catarina Island, southern Brazil, the bromeliad genus Aechmea comprises forms that combine an ornithophilous floral syndrome with relatively short corollas. Our aim was to assess and compare the effectiveness of bees as pollinators of syntopic A. lindenii, A. nudicaulis and A. caudata in relation to hummingbirds. The three species have similar, scentless yellow flowers. All are self-incompatible and, therefore, pollinator-dependent. Flowers secrete nectar throughout the day with sugar concentrations around 30%. Frequency of flower visitors was recorded throughout the day and pollinator effectiveness was measured as seed set per flower after single visits by either bird or bee visitors. Bees of 19 species (Apidae and Halictidae) visited the flowers. Bombus and Euglossa spp. were the most frequent visitors (40-56% of visits) and always more frequent than the visits by hummingbirds. Single visits by the hummingbird Thalurania glaucopis produced highest seed set in A. nudicaulis and A. lindenii, but failed to pollinate flowers in A. caudata. Among the bees, Bombus morio was the most effective pollinator of A. lindenii and A. caudata, followed by Euglossa and Xylocopa bees. In A. nudicaulis pollination effectiveness was highest for Xylocopa spp. and Augochlora spp. Except for A. caudata, bees were always much less effective pollinators than hummingbirds. We conclude that the pollination system in the three species is bimodal using different groups of pollen vectors. In spite of their ornithophilous syndrome bees play an important role in pollinating Aechmea flowers because low pollinator effectiveness per visit by bees is partly compensated by the high frequency of their visits. The redundancy in the pollination service provided by the use of different pollinator groups may assure seed set even in the absence of the proper pollinator as in the case of A. caudata.

3. Quantification of Pollen grains in honey. T. Blacquire, C. Hok-A-Hin, C. van

Dooremalen (Plant Research International, Wageningen University & Research, PO Box 69, 6708 PB Wageningen, the Netherlands)

The relative abundance of pollen grains of different plant species is generally determined in honeys to classify these with regard to botanical origin. In addition, the number of pollen grains per gram of honey are sometimes determined. Some specific honeys tend to have low numbers of pollen grains, while other honeys have high numbers. The number of pollen grains again can be used in classification. There is however very little information on

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the real pollen content of honeys, i.e. the amount of pollen, as g pollen / gram honey. Such information might be important when considering contamination of honey with allergenic pollen grains, or toxin containing pollen or genetically modified pollen species. Of local honeys, so far only five specimens, we determined the number of pollen grains per gram honey, using a Brker cytometric counting chamber. It was found that the number of grains ranged from 1 000 to 27 000 grains per gram honey. The counting method still needs to be improved since the results obtained depended on the final suspension volume before counting. To obtain calibration curves to relate pollen weight to pollen numbers, we spiked artificial pollen less honeys with a calibration range of pollen additions. We used five pollen species separately: Aceraceae, Brassicaceae, Fabaceae, Rosaceae, and pollen from Phacelia. After thoroughly mixing of the pollen through the honeys, the pollen was extracted from the honeys according to normal washing and centrifugation procedures, and the numbers were counted in a Brker counting chamber. We obtained statistically significant and strong correlations between added pollen (mg) and the numbers counted. The slopes of the lines represent the number of pollen grains per gram of pollen. It was found that pollen grains of Phacelia weighed 4.3 ng per grain, those of Aceraceae 67 ng/grain. A high pollen honey (10 000 grains/g) would thus only contain a few hundred g pollen per gram of honey.

4. Project Healthy colonies Does an improved consultation of beekeepers

lead to lower colony-losses? G. Brunnemann-Stubbe, V. Poker, R. Bchler (Landesbetrieb Landwirtschaft Hessen, Bieneninstitut Kirchhain, Erlenstrasse 9, 35274 Kirchhain, Germany)

Although there are effective preparations and treatment schemes, many colonies are still dying from the results of varroosis It is our aim to verify why those schemes are not complied with and how the consultation of beekeepers can be improved. 136 beekeepers with more than 1000 colonies from 4 beekeeper-societies in Hessen are taking part in this project. The most important part of the project are practical on-site demonstrations at the bee yards where all elements of an integrated treatment scheme against varroosis are presented and tried out by the beekeepers. It turned out that many beekeepers are unable to adapt given recommendations into their own practice because they are unaware of important connections. Bee samples out of 10 % of the colonies were examined in autumn in order to verify the health status. Nosema spec. as well as CBPV and SBV were scarcely detected. Infestation with Varroa destructor was 1.4 mites / 10 g bees (median). Bee samples in which DWV or ABPV had been detected were showing considerably higher rates of varroa as those without viruses. 75 % of the beekeepers used formic acid and oxalic acid or Perizin as a treatment against varroosis. During summer treatment with formic acid more than 30 different treatment methods were used which are to be distinguished by type of dispenser, concentration and amount of formic acid, duration, time as well as weather. We are unable to make beekeepers change their mind about the multitude of methods of treatment within a short time. Thus it is important to enable them to assess the health status of their colonies and the result of their treatment against varroosis in order to take necessary actions. When consulting beekeepers it is important to combine information directly with practice at the bee yard.

5. The serotonin 5-HT2 receptor system of the honeybee (Apis mellifera). W.

Blenau, M. Thamm, D. Rolke, A. Baumann (Institut fr Bienenkunde, Polytechnische Gesellschaft, Goethe-Universitt Frankfurt am Main, FB Biowissenschaften, Karl-von-Frisch-Weg 2, 61440 Oberursel, Germany)

Many physiological and behavioral processes in both vertebrates and invertebrates are controlled and/or modulated by the serotonergic system. In the honeybee Apis mellifera, serotonin has been implicated in division of labor, visual processing, and learning processes. A key issue to further increase the understanding of the serotonergic system is to unravel the molecular and functional properties of the corresponding 5-HT receptor subtypes. We have

characterized Am5-HT2 and Am5-HT2 which constitute the honeybee 5-HT2 receptor system. Both receptors share the common characteristics of G-protein-coupled receptors. Truncated transcripts representing alternative splice variants were detected for both 5-HT2

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receptors. HEK 293 cells were stably transfected with either the Am5-HT2 gene or the Am5-HT2 gene. Activation of both receptors with serotonin causes an increase in the intracellular Ca2+ concentration with Ca2+ being released from the endoplasmic reticulum in an IP3 dependent manner. To determine pharmacological profiles we tested the ability of a range of synthetic agonists to mimic the responses of the receptors to serotonin and the effects of synthetic antagonists to block the actions of serotonin Both receptors show a similar pharmacological profile with some subtle differences. The mRNA expression patterns were investigated by quantitative real-time PCR. Interestingly the expression of the full length variants of both honeybee 5-HT2 receptors is highest in the hypopharyngeal gland, whereas expression in the nervous system is very low for both receptors.

6. The loss of bees an important and puzzling parameter in population

dynamics of honey bee colonies. G. Liebig1, P. Aumeier2, O. Boecking3 (1 Landesanstalt fr Bienenkunde, Universitt Hohenheim, 70599 Stuttgart, Germany; 2 Ruhr-Universitt Bochum, Universittsstr. 150, 44801 Bochum, Germany; 3 LAVES, Institut fr Bienenkunde, Herzogin-Eleonore-Allee 5, 29221 Celle, Germany)

If not swarming, the development of honey bee colonies is characterized by a sequence of growth in spring, stagnation at high level in summer and shrinking in autumn and winter. For the accurate evaluation of colonies loss of bees, they have to be continuously monitored according to the Liebefeld method. Thus, the bee mortality in between two estimations can be computed by the difference of the bee potential (= sum of bees and [capped] brood cells) and the number of bees [12 or] 21 days later. During the breeding period (usually March to October) a total of maximal 250000 worker bees hatch in each colony. Nearly the same quantity of worker bees is lost during that period, equivalent on average about 1000 bees per day. Bee loss is highest in summer with about 2000 bees per day. Nevertheless colonies keep their strength, due to 2000 hatching bees every day. In winter, stronger colonies absolutely and relatively spoken lose more bees than weaker ones, on average 50 bees per day. However, the weaker the colonies are in October, the higher is the mortality risk during winter season. In fact, the daily bee loss cannot be monitored by the use of death traps, as dying bees usually leave the hive in a flying manner. Only cold winters offer the chance to find the dead bees at the hive bottom. During the breeding period, usually the loss of bees is positively and strongly correlated with the gain in bees. However, bee mortality is reduced if capped brood is removed, and increases after addition of capped brood, respectively. Yet, the loss of bees neither is affected by foraging activities, nor by processing liquid or compact food. Diseases as varroosis, nosemosis or chalk brood may affect mortality only if colonies are heavily infested. However, honey bee colonies are considerably much more susceptible to infestation with Varroa destructor during autumn and winter than during summer.

7. Biological control of Varroa destructor using entomopathogenic fungi. J.

Pflugfelder, P. Gallmann (Schweizer Zentrum fr Bienenforschung, Liebefeld Agroscope ALP, Schwarzenburgstr. 161, 3003 Bern, Switzerland)

The serious concerns about the current situation of Varroa destructor where chemical control offers only few substances all incurring risk of resistance in the mites prompted efforts in the development of a sustainable biological control based on fungal pathogens that have a low potential to generate resistance. We have tested several fungal strains in a high throughput screening bioassay for virulence to Varroa destructor and non-target effects to Apis mellifera workers. Mites were produced with a standard procedure and collected from emerging worker bees. Treatments were performed by immersing the mites in a 1x10 -7 spore solution of the strains. Minimum 10 replicates per treatment, each containing 5 mites kept in a small compartment were incubated at 34.5 C on white eyed worker pupae. Mortality and sporulation of the fungus was checked daily. The small compartments simulated the conditions in the brood cell resulting in an extended longevity of the mites compared to other test methods. The low mortality in the controls (10% at day 6) is crucial for the assessment of fungi. Among the tested strains we found only two candidates which killed

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the mites with an impressive speed (90% 4 days post treatment) while all the other strains did not differ from the control. The lethal time (LT50) was 3.04 and 2.78 days for two fungus strains and 9.96 days in the control treatment respectively (n=20). Further we could demonstrate a dose response and found no decrease in the biological activity of the spore solution after storage of one week. In all the tests the worker bees did not show any signs of fungus sporulation up to the adult stage. The strains have proved their potential as the most virulent fungi against Varroa destructor based on the LT50 data published. For practical use as a control we will develop a formulation for this promising strains and search for a biological distribution mechanism to deliver the fungus to the target.

8. Identification of first toxins of Paenibacillus larvae. A. Fnfhaus, L. Poppinga,

E. Genersch (Lnderinstitut fr Bienenkunde Hohen Neuendorf, Friedrich-Engels-Str. 32, 16540 Hohen Neuendorf, Germany)

American Foulbrood of honey bees is a notifiable epizootic of honey bee larvae caused by the Gram-positive bacterium Paenibacillus larvae. Larvae become infected by the ingestion of larval food containing P. larvae spores. The disease process can be divided into a non-invasive and an invasive phase. During the non-invasive phase, P. larvae spores germinate in the midgut and vegetative bacteria massively proliferate in the larval midgut lumen. The invasive phase is characterized by P. larvae attacking and penetrating the midgut epithelium and entering the haemocoel. Breaching of the epithelium is accompanied by a rounding-up of epithelia cells, a phenomenon known to be induced e.g. by proteases and toxins. Common bacterial toxins are ADP-ribosylating AB-toxins mostly targeting the actin cytoskeleton thereby destroying cell-matrix and cell-cell contacts within the host tissue. AB-toxins consist of an enzymatically active A-domain and one or more B-domains which mediate binding of the toxin to the host cell followed by translocation into the host cell. By sequence analyses we recently identified three putative ADP-ribosylating AB-toxins in the P. larvae genotype ERIC I, Plx1, Plx2 and Plx3. For functional analysis the according toxin genes were knocked out using the TargeTron Gene Knockout system and knock-out mutants were tested in exposure bioassays. In comparison to the parent wild-type strain the knockout mutants of Plx1 and Plx2 showed a marked reduction in larval mortality confirming the role of these putative virulence factors for pathogenesis of P. larvae infections.

9. CBP, a chitin-binding protein of Paenibacillus larvae, has an important role in

AFB pathogenesis. E. Garcia-Gonzalez1, A. Jakubowska2, E. Genersch1 (1 Lnderinstitut fr Bienenkunde Hohen Neuendorf, Molekulare Mikrobiologie und Bienenkrankheiten, Stolperstr. 40, 16540 Hohen Neuendorf, Germany; 2 Department of Genetics, University of Valencia, 46100 Burjassot, Spain)

American foulbrood (AFB) is considered the most contagious and destructive infectious disease in honeybees, caused by the Gram-positive, spore-forming bacterium Paenibacillus larvae. Despite the unquestionable impact of this disease, molecular mechanisms involved in pathogenesis still remain elusive. It has been shown that P. larvae spores ingested by young bee larvae proliferate massively in the midgut lumen and that breaching the epithelium is one of the last steps in the disease process. However, to achieve their way through the gut, bacteria must first penetrate the peritrophic matrix, a chitin-rich protective layer of the larval gut. Therefore, we hypothesized that chitin, the major component of the peritrophic matrix, may be a target for pathogens and that chitin-binding and chitin-degrading proteins play a major role in both attachment and local degradation of the peritrophic matrix. Here, we present our data on a chitin-bindin protein (CBP) secreted by P. larvae, which we identified as chitinase-like protein. Although this protein family has been traditionally described as chitin-binding protein it was recently shown that CBP21 from Serratia marcescens is an oxidative enzyme boosting the enzymatic breakdown of chitin by employing a different method than chitinases. We combined transcriptomic, proteomic and histological studies, both in vivo and in vitro, to functionally characterize CBP of P. larvae. We here demonstrate that CBP secreted by P. larvae has a similar way of action like CBP21 from S. marcescens and that it indeed plays an important role during P. larvae infection.

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10. Functional analysis of the virulence factor SplA, a Paenibacillus larvae ERIC

II specific S-layer protein. L. Poppinga1, B. Janesch2, A. Fnfhaus1, G. Sekot2, E. Garcia-Gonzalez1, G. Hertlein1, K. Hedtke1, C. Schffer2, E. Genersch (1 Lnderinstitut fr Bienenkunde Hohen Neuendorf, Friedrich-Engels-Str. 32, 16540 Hohen Neuendorf , Germany; 2 Department fr NanoBiotechnologie, NanoGlycobiology, Universitt fr Bodenkultur Wien, Muthgasse 11, 1190 Wien, Austria)

The etiological agent of the globally occurring epizootic American Foulbrood (AFB) is the grampositive bacterium Paenibacillus larvae (P. larvae), with the two differently virulent genotypes ERIC I and ERIC II, predominantly causing AFB outbreaks in Europe. In order to understand differences in pathogenesis we applied comparative proteomics and genomics to identify factors putatively associated with the observed virulence differences between the two genotypes. Analyses revealed that P. larvae ERIC II synthesises an S-layer protein, named SplA (S-layer protein P. larvae A), which might function as a putative virulence factor during bacterial infection. In our study we showed that in ERIC I genomic information of SplA is disrupted by a frameshift mutation. We could also demonstrate that SplA is a true S-layer protein by in vitro self assembly studies of recombinant, purified SplA. The protein assembles into a square 2D lattice detected by electron microscopy. To functionally analyse the proteins role during infection we successfully constructed a splA knockout mutant of P. larvae ERIC II strain 04-309 (04-309 splA) by a mobile group II intron mediated method (targetron). Adhesion capacity to primary pupal midgut cells was measured of 04-309 wildtype and splA deficient mutant cells, as well as differences in cell and colony morphology. Honey bee larvae were infected with P. larvae 04-309 (ERIC II) wildtype and knockout strain in in vivo bioassays. The outcomes clearly support the assumption of SplA representing a virulence factor during P. larvae ERIC II pathogensis. SplA is the first evident virulence factor for this species and our results clearly help us to understand genotypic differences in virulence of P. larvae.

11. Alternative antibiotics against nosemosis. S. Gisder, E. Genersch

(Lnderinstitut fr Bienenkunde Hohen Neuendorf, Friedrich-Engels-Str. 32, 16540 Hohen Neuendorf, Germany)

In vitro-infection models have become indispensable tools for the study of pathogen-host interactions on cellular or molecular level. With the help of these models host specificities, pathogen attachment, pathogen entry and propagation can be clarified. Furthermore, they are suitable for in vitro-screenings of potential effective substances against parasite infections. The obligate intracellular parasites Nosema apis and Nosema ceranae are important and virulent pathogens of the European honey bee causing dysentery in adult bees (called nosemosis). At the time being, Fumagillin is the only effective agent against nosemosis, however, the use of this antibiotic is not allowed in bees. Therefore, there is a demand on identifying novel anti-nosemosis substances. The recently established cell culture model for Nosema spp., based on the infection of the heterologous, permanent lepidopteran cell line IPL-LD-65Y, provides a previously unavailable means for studying pathogen-host interactions but also for developing substance-screening assays. We here describe the use of this model for the development of a medium-throughput screening system for the in vitro-screening of potential anti-nosemosis substances. Using (i) Nosema spp.-infected cell cultures, (ii) a newly developed, RT-PCR-based ELISA detection system for the expression of structural protein gene of Nosema spp., and (iii) Fumagillin as positive control we identified two new antibiotics, which showed the same performance as Fumagillin.

12. Effects of chronically administered sublethal thiacloprid doses on the

vitality of honeybee colonies. R. Siede, L. Faust, C. Maus, B. Grnewald, R. Bchler (Landesbetrieb Landwirtschaft Hessen, Bieneninstitut Kirchhain, Erlenstr. 9, 35274 Kirchhain, Germany)

Pesticides are thoroughly checked for side-effects on honeybees. Besides acute toxicity effects, long-term exposition might induce subtle impacts on colonies. This study

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aims to assess potential hazards of low doses of the neonicotinoid thiacloprid on full bee colonies under field conditions. Thirty colonies were started with young queens in July 2011 from shook swarms, divided in three groups with ten colonies each, and migrated to an experimental yard of Bieneninstitut Kirchhain. They were provided with sugar syrup containing either 200 ppb (T1) or 2000 ppb (T2) thiacloprid or syrup alone (control group K). Every 3 weeks the colonies were weighed, their amount of brood cells calculated from measurements of the brood area and their strength was estimated with the Liebefeld-technique. Dead bees were counted in entrance traps every third day. Honey and bee bread were sampled for analyzing residues of thiacloprid using LC-MS/MS. No differences in weight gain (p=0.46, glm, repeated measures, software spss), number of adult bees (p=0.634) and number of dead bees (p=0.098) were detected between the groups. By contrast, the number of brood cells differed (p=0.046): K-colonies generated 9976 brood cells, T1-colonies had 9161 brood cells and T2-colonies had only 8582 cells (means of all repeated measures). The relation between brood and bee number is a parameter of brood care: Mean values per bee were 0.24 for T2, 0.27 forT1 and 0.28 for K (differences not significant, p=0.123). These observations were obtained during the first year of the study. They may hint to differences in brood activity between the groups, but not to obvious adverse effects on colony survival or development. Since our project is scheduled for 3 consecutive years, replications performed in 2012 and 2013, should yield highly reliable observations on effects of long-term exposure to thiacloprid.

13. Residues of insecticides and fungicides in nectar and honey after using

different application technologies in blooming Brassica napus. K. Wallner, K. Schmitz (Landesanstalt fr Bienenkunde, Universitt Hohenheim, August-von-Hartmann-Str. 13, 70599 Stuttgart, Germany)

During the blooming phase rape is attacked by different pest species like pollen beetles and mould diseases. To prevent losses of yield, rape has to be treated with different pesticides during the blooming phase. Usually insecticide and fungicide mixtures are sprayed directly on the open rape flowers. These pesticides are collected by the bees together with nectar and pollen. Residues in honey and bee bread may cause discussions with consumers and beekeepers. In a field study with rape we tested an alternative application technology which only hits the rape plants below the bloom level. Similar under leaf technology is already used in vegetable farming (Dropleg). For comparison we treated 15 ha rape on one experimental farm conventional and on the other farm with the alternative technology. We used the insecticide Mavrik with the active ingredient tau-Fluvalinat and the fungicide Ortiva with the active ingredient Azoxystrobin. At both locations returning foraging bees were collected for a period of seven days twice a day at the hive entrance. The nectar out of the honey sacs and the pollen loads were checked for pesticide residues at the residue laboratory of the Apiculture Institute Hohenheim. Further we checked unripe honey extracted from honey combs and samples from the honey harvest at the end of the flowering period from both farms. After both kinds of application measurable residues were found in the collected nectar and pollen. But the nectar from the foraging bees of the Dropleg application had significant lower levels of both substances. Furthermore the extracted honey of the Dropleg hives had no measurable residues while the honey from the conventional application was contaminated in the expectable range.

Posters

14. Foraging and pollination behaviour of Apis mellifera adansonii Latr. (Hymenoptera, Apidae) on Gossypium hirsutum L. (Malvaceae) flowers at Dang (Ngaoundr, Cameroon) M. Sanda, F.-N. Tchuenguem Fohouo, D. Brckner (Forschungsstelle fr Bienenkunde, Universitt Bremen, FB 2, Postfach 330440, 28334 Bremen, Germany)

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To evaluate the Apis mellifera adansonii L. impact on fruit and seed yields of Gossypium hirsutum L., A. m. adansonii workers foraging and pollinating activities were studied in Ngaoundr during two seasons (August - September 2009 and August - October 2010). Observations were made on 240 flowers each year and divided in three treatments. Two treatments differentiated according to the presence or absence of protection regarding A. m. adansonii and other insects visits; the third protected and uncovered when flowers were open, to allow A. m. adansonii visits. Workers seasonal rhythm of activity, its foraging behaviour on flowers, its pollination efficiency, the fructification rate, the number of seeds/fruit and the harvested pollen. The mean foraging speed was 9.41 flowers/min in 2009 and 8 percentage of normal seeds were evaluated. Results show that A. m. adansonii foraged G. hirsutum flowers throughout the whole blooming period. This bee intensely and preferably.41 flowers/min in 2010. The fructification rate, the number of seeds/fruit and the percentage of normal seeds of unprotected flowers were significantly higher than those of flowers protected from insects. Through its pollination efficiency, A. m. adansonii provoked a significant increment of the fructification rate by 60.84% in 2009 and 36.48% in 2010, as well as the number of seeds per fruit by 94.23% in 2009 and 31.41% in 2010, and the percentage of normal seeds by 94.23% in 2009 and 33.49% in 2010. The installation of A. m. adansonii colonies close to G. hirsutum fields could be recommended to increase fruit, seed and honey yields, and pollen production as a hive product in the region.

15. Drift of Dust containing insecticides during sowing of maize- exposure and

effects on bees (Apis mellifera L.). P.-Th. Georgiadis, J. Pistorius, U. Heimbach (Julius Khn-Institut, Institut fr Pflanzenschutz in Ackerbau und Grnland, Messeweg 11-12 38104 Braunschweig, Germany)

For the investigation of effects of insecticidal dust drift on honey bees (Apis mellifera L.), two drift experiments were conducted under field realistic conditions during sowing of maize seeds treated with Clothianidin (Heubach - Value 2010: 0,86 g / 100000 seed with 10,6 % a. s. in dust; Heubach - Value 2011: 0,45 g / 100000 seed with 19,2 % a. s. in dust). Maize was sown by a pneumatic vacuum operated precision air planter with a drift reducing deflector. In 2010 the experimental area (flowering oil seed rape) was in the middle of two fields reserved for maize drilling depending on wind direction, in 2011 the drill area was surrounded by two areas with flowering oil seed rape. Flowering oilseed rape exposed to dust drift was used as treatment, OSR in opposite to the wind direction on the day of sowing was used as control. For each treatment, directly along the edge of the flowering oil seed rape 4 hives each for field exposure as well as three gauze-covered tunnel tents (16 x 6 m) with bee hives for the semi-field experiment were set up. Before sowing, bee hives in the tents were closed and the gauzes from the tunnels at the distance of 0 m to the drilling area were removed. Immediately after sowing, which took about 1 h, the tunnels were covered again and the hives reopened. Field hives were not closed and actively foraging. Foraging intensity, mortality, behavior was assessed in the trials for 7 days, brood and colony development up to 28 days after sowing. For exposure assessment samples of flowers, bees exposed in wire cages, petri dishes at 1, 3, 5, 10 and 20 m from the edge of the drill area as well as samples of nectar, pollen and dead bees analysed for residues. Wire cages with 10 bees each were set up at 1, 3, 5, 10 and 20 m distance from the field edge of the drill area. In both trials bee mortality was clearly increased, especially in semi-field conditions. The large amount of residue data enables a interpretation of the relative importance of different exposure routes and a refined risk assessment.

16. Particle size and toxicity of abraded seed coating? H. Mhlhuser, K. Wallner

(Landesanstalt fr Bienenkunde, Universitt Hohenheim, August-von-Hartmann-Str. 13, 70593 Stuttgart, Germany)

A new technology (Retsch CAMSIZER) was used to characterize the size of abraded dust particles of maize seeds used in the Rhine Valley in 2008. These particles are created during handling and transport of seeds in packages and during the sowing process in pneumatic drilling machines. Noticeable amounts were generated when the coating was

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done neglecting the instructions for a correct use of the product. Abraded dust is under further mechanical influence in the drilling machine, which may lead to an additional fragmentation of crude particles to fine dust. In 2008 dust particles of the Poncho pro (Clothianidin) coatings reached flowering plants and caused damage to bee colonies that were collecting contaminated nectar and pollen. Dust particles were detected in the mid gut of poisoned bees and in pollen loads from returning forager bees by microscopic analysis. The size of the particles was in the size range of pollen grains. Abraded Poncho pro dust particles of different size (< 100 m) were fed to caged single bees to determine their size dependent toxicity. Due to the extreme insecticidal potential of Clothianidin already very small particles, which are expected to be more influenced by wind and thermal lift than by gravity, showed poisoning effects on the bees. With regard to these results it is important, to improve seed dressing not only with regard to the reduction of abraded dust but also to avoid the release of weightless fine dust particles during the seeding process.

17. A method for testing effects of honey components on bee development. B.

Lichtenberg-Kraag, E. Genersch (Lnderinstitut fr Bienenkunde Hohen Neuendorf, Friedrich-Engels-Str. 32, 16540 Hohen Neuendorf, Germany)

Climate changes and mans interventions in the ecosystem influence both flora and fauna. While new plants as nectar sources are available for our domestic bees, others disappear, resulting in a change of honey composition. These changes raise the question for beekeepers whether or not all components of nectar or honey will provide a good enough nutritional basis for their colonies during winter. In vitro rearing of larvae has been used in the past for analyzing host-pathogen interactions or caste determination or larval physiology. We here present our data obtained with a modified larval rearing assay adopted for testing the nutritional value honey of different floral sources has for larvae. The sugar components of the larval food were substituted by unifloral honey from Brassica sp. and Robinia pseudoacacia. The course of larval and pupal development was recorded and final size and fitness of the emerging adult bees was analyzed. No effect on the development from larvae to bees depending on honey source was observed. About 60% of the larvae matured to normal bees independent of the carbohydrate source of the food. Using this method, the influence of honey from definite nectar sources on larvae development can be investigated. Honey which shows significant effects on larval/pupal development will be further analyzed in detail to identify potential beneficial or adverse components.

18. Characterization of regional honey as a reflection of the occurring flora of

an area. E. Etzold, B. Lichtenberg-Kraag (Lnderinstitut fr Bienenkunde Hohen Neuendorf, Friedrich-Engels-Str. 32, 16540 Hohen Neuendorf, Germany)

Biosphere reserves can be classified as unique according to landscape and floral diversity. In certain areas local trademarks have already been established. Beekeepers can use the logo of the biosphere reserve for the marketing of their honey. Local trademarks support the identification of the consumers with natural and cultural landscapes and assist them in orientation for proven quality. The demands on quality of such regional products are usually higher than required by legislation. This increases the image of the area. The authentification of honey is based on the microscopic pollen analysis which gives information on both, the botanical and the geographical origin. In combination with quality analysis honey profiles typical for different areas can be generated. In this project, the biosphere reserve Spreewald was characterised with respect to landscape and flora for 4 years. The honeys from the area were organoleptically and physico-chemically analysed. The pollen spectrum was determined. Based on these investigations the typical floral sources of these honeys were inferred. The profile of the Spreewald area was determined. Especially in 2010 and 2011, a high number of samples generated representative results. In contrast to an urban region like Berlin the main honey flow in the Spreewald area is characterized mainly by wildflowers with trees and bushes being less represented. The pollen diversity is high. As expected, the fraction of nectar from Brassica sp. is higher than in the Berlin region. Based on the data of the project, the label Spreewlder Stiftungshonig was created by the

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Brgerstiftung Kulturlandschaft Spreewald e.V.. This provided support to local beekeepers in regard to the marketing of their honey.

19. A colony based drone keeping method allows analysis of sperm

characteristics in honey bee drones. C. Vogt, N. Hcherl, S. Hrtel (Lehrstuhl fr Tierkologie und Tropenbiologie, Universitt Wrzburg, Biozentrum, Am Hubland, 97074 Wrzburg, Germany)

Drones are essential for the survival of a honey bee colony because of their role in reproduction. We conducted research on experimental factors that influence the maturity of drones and characteristics of sperm production. Newly emerged drones were kept using three different methods. (1) single queen cages which allowed only little movement and in which drones were supplied by nurse bees,. (2) comb cages in the bee hive providing the ability to walk but not to fly. (3) bee hives in which drones were allowed to roam freely within one compartment of the hive, with the possibility to fly on day16 after hatching. Our data prove that the production of sperm is strongly linked to the ability of drones to move freely during their reproductive development. The number of drones producing sperm increased significantly from small queen cages over comb-cages to hives. In a preliminary ecotoxicological study, drones showed a tendency towards decreased survival after feeding on sub-lethal doses of imidacloprid or dimethoate. In comparison to drones that were fed pure sugar solution, pesticide effects on sperm production were not observed. Further research is needed to analyse the risk of sub-lethal doses of pesticides on the reproductive performance of drones.

20. Effects on honey bee colonies (Apis mellifera L.) following a granular

application of Santana containing the active ingredient clothianidin in maize in 2010 and 2011. I. Illies1, S. Berg1, J. Pistorius2, G. Bischoff3 (1 Bayerische Landesanstalt fr Weinbau und Gartenbau, Fachzentrum Bienen, An der Steige 15, 97209 Veitshchheim, Germany; 2 Julius Khn-Institut, Institut fr Pflanzenschutz in Ackerbau und Grnland, Messeweg 11/12, 38104 Braunschweig, Germany; 3 Julius Khn-Institut, Institut fr kologische Chemie, Pflanzenanalytik und Vorratsschutz, Knigin-Luise-Str. 19, 14195 Berlin, Germany)

Wireworms, which are the larvae of click beetles (Family Elateridae) have become a serious pest of corn. Therefore, the application of Santana, a granulate with the active ingredient clothianidin, was allowed in 2010 and 2011 under strict regulations in Germany. The granules are deposited in the soil during sowing, together with the grain of seed. Clothianidin belongs to the group of neonicotinoids and is toxic for bees. An exposure of honeybees to clothianidin, either by dust during sowing or through the guttation liquid of the plant might be possible. When guttation liquids collected with the beginning of guttation were mixed with sugar and fed to caged bees high mortality was observed; bees fed with this mixture died within one hour. Thus, the effect of guttation under realistic field conditions was observed. In 2010 and 2011 honey bee colonies were placed at fields before the sowing of corn and the application of Santana. To estimate the mortality of honey bees in the hive all colonies were equipped with traps for dead bees in front of the hive. In both years no increased mortality was recorded during sowing or the following days. During the guttation period the mortality observed in the bee traps increased marginally over a few days. In some samples clothianidin was detected. No negative effects on colony development were recorded in either of the years 2010 and 2011.

21. Reproductive Biology and Flower Visitors of the Wild Service Tree (Sorbus

torminalis L. CRANTZ). J. Werres, D. Wittmann (Rheinische Friedrich-Wilhelms-Universitt Bonn, Institut fr Nutzpflanzenwissenschaften und Ressourcenschutz, Abteilung Tierkologie, Melbweg 42, 53127 Bonn, Germany)

The Wild Service Tree (Sorbus torminalis) is a very rare and only locally distributed fleshy fruited tree. For species conservation targeted pollination management strategies are necessary. In Germany, S. torminalis is often is a relic of the historical coppice forestry

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because the species needs light and is competitively rather weak. Today there are only some scattered and mostly isolated populations left which are endangered by the decreasing diversity of their genetic material. Furthermore, the gene flow by pollination between populations and even individuals is extremely low. Pollination is the only way to conserve the gene pool of these populations and to produce seeds for forestry purposes. Despite this situation, there are no studies about the pollinators and flower visitors of S. torminalis. Self incompatibility has been described in the literature, but the data about this topic are ambiguous. In order to test for self incompatibility and the quantities of fruit- and seed-development, four pollination treatments were carried out. Additional research was undertaken on the insects visiting the flowers. They were trapped with an insect net. The field experiments were carried out in a forest stand in a nature reserve close to Bonn and a seed orchard between Cologne and Bonn. In total 42 species of flower visitors were detected. The first results of the pollination experiment indicate that seed development in S. torminalis is highest when flower visitors have free access to the flowers (average 3,9 seeds of 5 ovules). The tests for apomixis and geitonogamy show that both types of reproduction are possible but that they result in a lower number of seeds/fruit (averages 1,3 and 1,6 seeds/fruit) . The abortion rate in every treatment is rather high (72-96 %). This is caused by sucking aphids (Homoptera) on the one hand while on the other hand it does not seem to be unusual for S. torminalis.

22. Residues in Guttation droplets- potential risk for honey bees (Apis mellifera

L.)? Field trials with set up of bee colonies at field edges of seed treated Winter oilseed rape and cereals. Ina Joachimsmeier, Jens Pistorius, Detlef Schenke, Wolfgang Kirchner (Julius Khn-Institut, Institut fr Pflanzenschutz in Ackerbau und Grnland, Messeweg 11-12, 38104 Braunschweig)

As bee colonies seem to prefer water sources in the near surroundings of the hive, a field trial was set up to investigate whether bees prefer guttation drops as a source for water compared to dew or rain drops. Furthermore it was investigated if bees still use these water sources up to 50m away from their hives. The experiment was conducted from 10th of April until 09th of May 2010 and was set up on one organically and one conventionally managed field near Ahlum (Lower Saxony, Germany). The experimental fields consisted of two plots; one planted with cereals and one adjacent plot with oilseed rape. In the organically managed variant the seeds were untreated. While in the conventional variant the cereal seeds were treated with a fungicide (Zardex G, a.s. Imazalil), the rape seeds were treated with an insecticide (Elado, a.s. Clothianidin). On each field a total of three one-room and two two-room bee colonies (Zander) were set up. The bee hives were placed in the cereal plot with a distance of 0m* (field border), 10m, 20m, 30m and 50m* to the adjacent oilseed rape field and a distance of 50m from each other (*two-room bee colonies, entrance pointed toward the oilseed rape plot). During every daily assessment the climatic conditions, the growth stage of the crop plants and the presence of guttation, rain or dew drops in both plots were recorded. In addition, the population development of the beehives, the daily bee mortality and with the beginning of bee flight, behaviour observations on honey bees interacting with plants were assessed. These observations were conducted several times daily until no more guttation droplets were visible at three fixed observation points for every bee hive (two points next to every the bee hives, one at the transition zone between the two plots). If no guttation was present, the observation was conducted only once. During the experiment 147 bees were observed scanning the surface of the plants without landing, 13 bees that took up guttation fluid and 36 bees which collected dew or rain drops. Some bees were found collecting guttation fluid 50m away from their hives but most were observed adjacent to it. Furthermore, in some dead bees residues of the seed coating were detected (Imazalil 0,11 - 329 ng/bee; LD50= 35 100 ng/bee)

23. Ionic current modulation of honeybee mushroom body and antennal lobe

neurons. S. Himmelreich, B. Grnewald (Institut fr Bienenkunde, Polytechnische

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Gesellschaft, Goethe-Universitt Frankfurt am Main, Karl-von-Frisch-Weg 2, 61440 Oberursel, Germany)

The honeybee (Apis mellifera) is a model organism for the study of learning and memory formation and its underlying cellular mechanisms. The neuronal pathway of the associative olfactory learning includes two neuropils: the antennal lobes (AL) and the mushroom bodies. Here, the excitatory cholinergic and octopaminergic reward pathways converge. For learning-related plasticity the coincident occurrence of the conditioned stimulus and the reward has to be detected. Two cellular coincidence detectors are possible: (1) The activation of a Ca2+-dependent kinase which may be activated by an elevated intracellular Ca2+-concentration induced by the simultaneous activation of the excitatory nicotinic acetylcholine receptor (nAChR) and an -adrenergic-like octopamine receptor (OAR). (2) The activation of an adenylyl cyclase-activated PKA which is activated by cAMP, a second messenger coupled to a -adrenergic-like OAR. Both hypotheses would lead to modulations of ACh-induced ionic currents. We performed patch-clamp recordings and cell Ca2+-imaging to investigate the cellular interactions of the nAChR and OAR of pupal AL neurons and KCs in vitro. The Ca2+-imaging showed that OA (10M) evoked Ca2+-transients in both AL neurons and KCs. These transients were blocked by mianserin (100M). Co-applications of ACh (500M) and OA, or serotonin and forskolin (all 10M) reduced the OAR-dependent Ca2+-signals. Patch-clamp experiments revealed decreased ACh-induced currents when co-applied with OA (100M) or serotonin (10M) and co-applications of ACh and forskolin (10M) reduced the ACh-current amplitudes in AL neurons. This indicates involvement of a cAMP-dependent signaling cascade. Our hypothesis of two coincidence detector mechanisms may be confirmed by these results. However, further experiments are under way to unravel the cellular bases of learning related synaptic plasticity.

24. Apis florea colony surface temperature changes in response to disturbance.

N. Haddad1, A. Batainha, J. Tautz2, S. Fuchs3 (1 National Centre for Agricultural Research and Extension (NCARE), Amman, Jordanien; 2 BEEgroup, Biozentrum, Universitt Wrzburg, Am Hubland, 97074 Wrzburg, Germany; 3 Institut fr Bienenkunde, Polytechnische Gesellschaft, Goethe-Universitt Frankfurt am Main, Karl-von-Frisch-Weg 2, 61440 Oberursel, Germany)

Open-nesting Apis florea are exposed to environmental threats, in particular to disturbances by predators. Bees on the nest surface have developed at least three different responses, termed hissing behaviour, body shaking and rearing-up behaviour which are elicited by concussions, optical stimuli or air gusts, respectively. We show here, by use of thermographic video recordings, that these behavioural responses are accompanied by substantial nest surface temperature elevations. Nests disturbed in either of these ways show increased surface temperature spreading from the centre of the disturbance. At ambient temperature of 20C mean surface temperature raised from 25.5C to 28.8C, but reached 30.3C in core areas of disturbance. In single workers, thorax temperature reached up to 40C. This temperature buildup took about 30 sec after disturbance, and ceased again after approximately 5 min. The main source of initial surface temperature increase comes from deeper and warmer levels of bees being exposed by the commotion of the outmost layer. Accordingly, excited workers showing high thorax temperatures in the range of 35 C and running over the nest surface emerge from the inner colony levels in already heated-up condition, rather than originating from the outer level whose bees would need to heat up first. However, the mantle bees themselves also start to warm up over time and at peak disturbance most surface bees express an elevated temperature of 29C, and are thus ready for defensive action.

25. For drones, the serious side of life begins at day six. N. Hirtz, D. Ahrens, H.

Gtschenberger, N. Hcherl, M. Pahl, J. Tautz (Universitt Wrzburg, BEEgroup Zoologie II, Am Hubland, 97074 Wrzburg, Germany)

The life-task of drones in a honeybee colony (Apis mellifera) is to produce sperm and to mate with a virgin queen. The drones role in the hive, however, is still a matter of dispute.

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It has already been observed where drones linger in the hive, including their ability to consume food by themselves even though they have a short proboscis. In this experiment, nutrition and spatial distribution of drones in the colony were tested, with emphasis on age-related variation. Videos were recorded in order to analyze the trophallaxis frequency, as well as the feeding behavior of drones and workers. Photographs served as recording of the drones spatial distribution in the hive. A total of 75 drones in three independent replicates were investigated. One to three day old drones were almost exclusively fed by workers (mean 94 % 3). During this time, they mostly stood on the brood comb (mean 85 % 7), because in this area, the chance to be fed by a nurse bee is higher. When the drones got older they were fed less often and began to take honey out of the cells on their own. At the age of six days, 82 % of drones were entirely independent of being fed by nurse bees. As soon as they began to feed themselves, they left the brood comb more often. The most preferred area of drones older than seven days was the edge of the comb near the nest entrance. Our results show, that trophallaxis and spatial distribution are associated with the drones age.

26. Apis mellifera sperm cells shrink during storage. J. Wegener, K. Bienefeld

(Lnderinstitut fr Bienenkunde Hohen Neuendorf, Friedrich.-Engels-Str. 32, 16540 Hohen Neuendorf, Germany)

Despite recent progress, the mechanisms by which honey bee sperm are kept alive and fertile for years within the spermathecae of queens remain mysterious. One way of elucidating these mechanisms could be to study the changes sperm cells undergo during storage. Recently, changes to the proteome of the stored spermatozoids and to their capacity to engender viable offspring have been evidenced. In the present experiment, we were interested in the effect of storage inside the spermatheca on sperm length. To study such effects, we evaluated the length of freshly-ejaculated sperm as well as that of cells stored by queens for between three and >24 months. In total 1129 cells from 64 samples were photographed and measured on the computer screen. The results show a slight but significant reduction of sperm length between freshly ejaculated semen and semen after three months inside the spermatheca (Mann-Whitney test; total n=28; P=0.002). During storage, a further steady reduction of sperm length was observed (Jonckheere-Terpstra-test; total n=55; P=0.02). The overall reduction of sperm length between unstored semen and semen from queens > two years old was 3.5%. As the shortest cells extracted from old queens were shorter than any cells found in freshly ejaculated semen, it seems likely that the reduction of sperm length is due to shrinkage of all sperm and not to the selective remainder/survival of shorter sperm inside the storage organ. The causes for this shrinkage are elusive. Possible explanations could be a contraction of strucural elements due to a change in electrochemical interactions with factors within the thecal lumen, or the partial digestion of cellular components by enzymes from either the sperm or the queen reproductive tract.

27. Influence of social cues on decision making of honeybees in a temperature

gradient. S. Hahshold, M. Szopek, G. Radspieler, R. Thenius, T. Schmickl, K. Crailsheim (Institut fr Zoologie, Karl-Franzens-Universitt Graz, Universittsplatz 2, 8010 Graz, Austria)

Experiments showed that young honeybees have a temperature preferrendum near 36C. In a circular temperature arena with a 2-dimensional thermal gradient only a minority of single young honeybees stay at their preferred temperature. Interestingly however, groups of bees locate themselves at 36C. Former studies in a complex gradient showed that these groups are also able to distinguish between an optimum (36C) and a sub-optimum (32C) and choose their preferred temperature. We conducted experiments to test the influence of a social gradient on the cooperative thermotaxis in honeybees. We introduced young bees into a temperature gradient and also into a social gradient in a circular arena. The temperature gradient was generated with heat lamps and the social gradient was generated with bees inside an acrylic cage with fence walls at the sub-optimum (32C). As a control an empty cage was placed at the optimum (36C). We investigated two different group sizes: 6 (n=11)

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and 24 (n=13) bees. Whereas in 25% of the trials with groups of 6 bees without caged bees the bees chose the optimum collectively, in trials with caged bees only in 9% of the trials the optimum was chosen. Whereas in 100% of the trials with groups of 24 bees without caged bees the bees chose the optimum collectively, in the trials with caged bees only in 30% of the trials the optimum was chosen. In 9 % (for 6 bees) and 30% (for 24 bees) of the trials the bees decided to aggregate in the sub-optimum. In trials without caged bees the groups never chose the sub-optimum whereas in trials with caged bees the sub-optimum was chosen collectively in 9 % (6 bees) and 30% (24 bees). With our experiments we could show that a social gradient influences the aggregation behaviour of young bees stronger than a temperature gradient.

28. Do artificially reared bees (Apis mellifera carnica) have the ability to nurse?

K. Tschigg, U. Riessberger-Gall, K. Crailsheim (Institut fr Zoologie, Karl-Franzens-Universitt Graz, Universittsplatz 2, 8010 Graz, Austria)

Artificial rearing of honey bee larvae in the lab became common in honeybee science nowadays. The desired result is to obtain well developed individuals. We investigated the behaviour of artificially reared honeybees using a two frame observation hive. Behavioural parameters like brood caring activities were regularly monitored. This is a remarkable phenomenon considering that artificially reared bees never received brood care by nest mates themselves during their own larval development. To verify the ability of nursing we compared the development of hypo-pharyngeal glands of artificially reared honeybees with those of naturally reared sisters. The rearing method of Aupinel et al. (2005, Bulletin of Insectology 58) was used. Bees were marked after emerging and introduced in a colony. Samples were taken after 6, 9 and 12 days. We measured the acini diameters and head fresh weights of 12 individuals per group and age. In all reviewed age-classes the head fresh weight was significantly higher in naturally reared bees (P

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(~53 %) while wall following, goal finding and immobility evenly contribute to the remaining 47%. Random walking is also the most consistent behaviour. In 18% of our observations random walkers exhibited this behaviour in all of four consecutive experiments. This value is about three times as high as for wall followers and goal finders. Additionally, we were able to correlate each behavioural class with its members resting behaviour and their probability to be found at specific zones of the arena. This evaluation of class specific behaviour is a crucial prerequisite for our future research plans.

30. Residues in Guttation droplets - potential risk for honey bees (Apis mellifera

L.)? A worst case semi-field scenario in maize with special consideration of impact on bee brood and brood development. M. Frommberger, J. Pistorius, A. Schier, I. Joachimsmeier, D. Schenke (Julius Khn-Institut, Institut fr Pflanzenschutz in Ackerbau und Grnland, Messeweg 11-12, 38104 Braunschweig, Germany)

In two worst- case scenario semi-field studies the possible risk of guttation for honey bees (Apis mellifera L.) was investigated. The study was performed under semi-field conditions, in maize treated with Clothianidin and untreated maize. The tents (4 treatment and 2 control) had an area of 96m2 (16 x 6m) each and were covered with a gauze permeable for wind and rain but impenetrable for bees. The study was repeated twice, in the first run (BBCH 13-15) with two variants, one with and one without artificial water source containing uncontaminated tap water. In the second run (BBCH 15-19) all variants had an artificial water source. The mortality of bees was assessed in dead bee traps and on linen sheets in the crop. The flight activity and behavior of bees at the entrance of the hives and in three flight squares in the crop were determined once daily. The observation time in the tents was 11 (1st run) and 10 (2nd run) days, the observation of brood development of 100 brood cells per hive was conducted nearly for four weeks following the protocol of Schur et al., 2003. During the whole observation period the occurrence of guttation was documented and, if guttation occurred, guttation droplets were sampled daily for residue analyses. Due to the weather conditions in the first run guttation was only observed once, which caused a high mortality in the waterless variation as well as a brood-termination rate of up to 100%. In the second run guttation of the corn occurred on 6 of 11 days. The mortality in treated and control variants was on a similar level and within normal range. In the first run of the study, in the artificial worst-case situation without any additional water supplies a high impact on mortality and also on the brood development was observed, indicating the sensitivity of the test system but representing an unrealistic worst case scenario. In variants with treated maize and additional water supply, no effects on adult mortality and brood were observed.

31. The long road of hyperthermia in control of varroa. W. Engels (Universitt

Tbingen, Institut fr Evolution und kologie, Auf der Morgenstelle 28, 72076, Tbingen, Germany)

Already 25 years ago Peter Rosenkranz studied thermopreference in Varroa mites and brood nest temperatures in honeybee colonies. Because the thermoresistance of reproducing female mites is lower than that of honeybee brood, hyperthermia as a measure for biotechnical Varroa control is possible. The optimum parameters had been programmed in a special incubator named Apitherm machine. This equipment made of stainless steel was on the market for one decade. The required alternating current of 220 V, together with its weight and size were handicaps for operations in the field, for which we had to co-transport a power generator on a trailer. Between 1988-1993 about 50 of our 200 colonies received only hyperthermia treatment, combined with drone brood removal We applied hyperthermia during the spring cherry blossom and again after the honey harvest in early summer. If increased mite fall indicated alarming colony infestation, an additional treatment was done in late summer or autumn. These measures had considerable success, as the experimental colonies did as well as the controls. We also tried to realize hyperthermia without complicated machinery in empty super boxes. Small heating devices were placed over the space between combs. But a uniform temperature level was not achieved by these constructions. In order to become independent from electric heating, we tested the use of

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solar energy using various panels and techniques. In Brazil experiments with tents were performed, however, sometimes even cooling was necessary. Only recently hyperthermia revival efforts were undertaken in Austria. Last year the Varroa Controller was presented, making use of our treatment program. Hyperthermia could be of interest especially for sideline beekeepers. The long road towards biotechnical Varroa control without acaricides and following mite resistance has approached a new station. The method could perhaps also be of interest in tolerance breeding programs.

32. Preliminary results of honeybee winter losses in Austria in 2010-2012.

H.Kglberger, I. Derakhshifar, R. Moosbeckhofer (Institute for Seed and Propagating Material, Phytosanitary and Apiculture, Agentur fr Gesundheit und Ernhrungssicherheit, Spargelfeldstrae 191, 1220 Wien, Austria)

A study aiming to clarify reasons of honeybee winter losses in Austrian apiaries has been carried out since the hibernation period 2010/2011. Beekeepers with affected apiaries have been invited to provide bee- and brood samples and to fill in a questionnaire concerning details about their beekeeping practice and their measures against Varroa destructor. In 2010/2011 42 cases were reported and investigated. In the ongoing hibernation period 2011/2012 33 cases of winter losses have been reported by 10 February 2012. Bee samples were tested for Nosema spores microscopically and subsequently positive samples for DNA of N. apis and N. ceranae by PCR. Furthermore bee samples were checked for presence of viral RNA of 7 viruses (see below) by PCR. Infestation by V. destructor was visually investigated in bee- as well as brood samples. In 2010/2011 N. ceranae (32 %), N. apis (3 %; both: n=79), ABPV (26 %), BQCV (40 %), CBPV (7 %), DWV (81 %), SBV (18 %), KBV (3%) and IAPV (4 %, all viruses: n=73), V. destructor in bees (66 %, n=82) and V. destructor in brood (90 %, n=31) were detectable in tested samples. In 2011/2012 ABPV (58 %), BQCV (38%), CBPV (0 %), DWV (88 %), SBV (33 %), KBV (8 %), IAPV (0 %) all viruses: n=24), V. destructor in bees (90 %, n=49) and V. destructor in brood (100 %, n=35) were tested and detectable as given in brackets. Most brood samples showed high infestation rates in the examinated brood cells and moreover multiple V. destructor mites per cell. High infestation rates of V. destructor and high prevalence of DWV are presumably main reasons for losses.

33. Effects of pollen diets on the longevity and development of hypopharyngeal

glands of honeybees (Apis mellifera L.). E. Blum1, I. Illies2, J. Tautz1 (1 Universitt Wrzburg, Am Hubland, 97074 Wrzburg, Germany; 2 Bayerische Landesanstalt fr Weinbau und Gartenbau, Fachzentrum Bienen, An der Steige15, 97209 Veitshchheim, Germany)

Three colonies of honeybees (artificial swarms with 3000 worker bees) without any storage of honey or pollen were fed with three different pollen diets: Feedbee, a commercial pollen substitute, maize pollen (hand collected) and mixed pollen (bee collected). The colonies were placed in outdoorflightcages contained no flowering plant, so the bees foraged exclusively on a feeder with sucrose solution. The respective pollen diets were administered ad libitium from feeding devices in the bottom board of each hive. A fourth colony (free foragers) was placed outside the flight cage and was able to collect pollen and nectar. Brood-rearing was measured by the Liebefelder method and all frames containing sealed brood were removed from the hive and placed in an incubator. The emerging young bees were picked up and caged by 20 worker bees per cage. A total of 10 cages per diet were observed for 28 days, mortality and food consumption daily recorded. Additional cages were filled to prepare and measure the hypopharyngeal gland 3, 6, 15 and 22 days after emerging. The colony outside the flight cage and the colony fed with mixed pollen reared about 1600 worker bees, followed by the maize pollen colony (620 bees) and the Feedbee colony (400 bees). Caged bees fed with Feedbee showed a significant higher mortality during the experiment (U-Test; N=10; p=0,011). The volume of hypopharyngeal glands of bees fed with mixed pollen decreases from day 3 to day 22. Bees fed with Feedbee and maize pollen showed a varied development of hypopharyngeal glands during the experiment. On day 15 and 22 the volume of hypopharyngeal glands of bees fed with Feedbee and maize pollen increased.

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34. Analysis of oxalic acid distribution in colonies of Apis mellifera by

computed Tomography. E. Rademacher1, A. Fahlberg2, M. Harz1 (1 Freie Universitt Berlin, Institut fr Biologie/Neurobiologie, Knigin-Luise-Strae 28/30, 14195 Berlin, Germany; 2Tierrztliche Klinik fr Pferde Seeburg, Germany)

In hive application of oxalic acid is registered to be conducted via trickling a defined dosage dissolved in sugar water depending on the colony size. The distribution is primarily achieved by bee-to-bee contact and not through trophallactic interaction. The efficacy of an agent mainly depends on its distribution. We used a macroCT scanner to visualize in hive distribution of oxalic acid. 2D images were taken from a nucleus colony. With a helical scanner a distance of 250mm was examined to make sure that the colony was captured completely. CT-images were reconstructed with 2mm slice thickness and the density of individual bees was measured in Hounsfield Units (HU). In a defined area of 100cm2 in the central area of the comb as well as the boundary area of the bee cluster the density of single bees was quantified over three combs (n=144). Imaging took place before application of oxalic acid (= control) and ten minutes afterwards. Only bees placed parallel to the marcoCT sectional plane were evaluated. Density values were transformed into ranks of 1 to 5 (1=low density of -400 to - 300 HU, 5=high density of 0 to +100HU). Visibility was achieved by adding the contrast agent Unilux (Iopamidol, 370mg Iod/ml, dosage 0.5l/bee) to the oxalic acid solution. Unilux was proven nontoxic to bees prior to the scanning experiments. The mean density value for the control measurement was -219.77 HU (SD 93.37) reaching a mean ranking value of 2.3. Ten minutes after the application the mean density reached -98.97 HU (SD 87.06, mean ranking of 3.5) which results in a significant difference to the control measurements (t-test, p 0.001, f = 286). The density of bees in the central part of the comb (-96.03 HU, SD 87.93) was very close to the density in the boundary areas (-101.92 HU, SD 86.7). These results demonstrate an even distribution of oxalic acid within a short reaction time of ten minutes.

35. Short and sweet - optimal performance of formic acid-based treatment in

honey bee colonies. P. Beinert1, P. Aumeier1, G. Liebig2, C. Lffler3, H. Aupperle4, W. H. Kirchner1 (1 Ruhr-Universitt Bochum, Universittsstr. 150, 44801 Bochum, Germany; 2

Landesanstalt fr Bienenkunde, Universitt Hohenheim, 70599 Stuttgart, Germany; 3

Universitt Leipzig, 04109 Leipzig, Germany; 4 Laboklin GmbH & Co KG, 97688 Bad Kissingen, Germany)

In honeybee colonies, effective treatment of Varroa destructor reduces winter

collapses significantly. Formic acid (FA), with its unique ability to kill both phoretic and

reproductive mites simultaneously, is the crucial device of successful management practices.

However, due to its narrow therapeutic index and the extreme variable efficacy depending

on ambient microclimatic and within-hive conditions, the mode of application has to be

carefully tuned for the optimal effect. In field trials we tested 12 different types of FA-

application in a total of 164 colonies (Liebig-Dispenser: LID; Mite away quick strips: MAQSTM;

Nassenheider horizontal: Nasshor; Nassenheider vertical with or without heater: Nassvert).

Treatments were carried out once or twice at apiaries in North Rhine-Westphalia and

Saxony-Anhalt, Germany, from August till October 2011. Population development and thus

bee and brood mortality was monitored according to the Liebefeld method and death traps.

Consistant and high efficacy was accomplished by use of 85% FA in the LID (81.918.8%

mite mortality) as well as the Nasshor (78.519%), respectively. The effect of 60% FA ranged

between 5.4 and 98.9%. Good performance of 60% FA could be generated with heated tiles

(Nassvert+heater: 67.821.1%) or huge wicks (Nasshor, 79.713.8). Long-term treatments

with low daily release of FA (Nasshor 60 or 85%: 18ml/d for 14d) caused a lower acute

mortality in adult bees, but higher brood damages compared to the short-term-treated groups

(LID: 25ml/d for 3d, 85%FA; MAQSTM: 70ml/d for 7d). In contrast to 14-day-applications, the

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short and heavy treatments never generated queen or colony losses, but stronger winter

colonies (LID and MAQSTM compared to Nasshor 60% or 85% and Nassvert: p

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38. Potential siderophore in Paenibacillus larvae. G. Hertlein, E. Garcia-

Gonzalez, L. Poppinga, A. Fnfhaus, E. Genersch (Lnderinstitut fr Bienenkunde Hohen Neuendorf, Friedrich-Engels-Str. 32, 16540 Hohen Neuendorf, Germany)

Bees are the most important commercial pollinators, used to increase the yield of crops depending on insect pollination. In addition, they pollinate flowering wild plants and thus contribute to the maintenance of many ecosystems. As any organism, bees are haunted by diseases and parasites. An especially dreadful disease is American Foulbrood, caused by the gram-positive spore forming bacterium Paenibacillus larvae. This highly contagious and fatal bee pathogen affects the brood of honey bees and hence has severe impact on the whole colony. P. larvae proliferates in the midgut of bee larvae after ingestion of spores, lives like a commensal until the whole cavity is filled before it breaches through the epithelium and thereby kills its host. Eventually, the cadaver is decomposed to a ropy mass. As the entire vegetative stage proceeds inside the larvae, all micronutritions have to be stolen from the host the most valuable among them being iron. Other pathogenic bacteria (e.g. Bacillus anthracis) established methods for extracting iron from the host proteins using siderophores. These are powerful iron chelators, specifically secreted in response to iron deficiency. Comparative genome analysis revealed that P. larvae harbours a gene cluster coding for a nonribosomal peptide synthetase (NRPS), which might produce a siderophore. First results of in vitro and in vivo experiments to determine the role and nature of the NRPS are shown. Knock-out of the NRPS cluster did not reduce virulence in vivo, did however alter swarming behaviour of P. larvae. The identity of the siderophore is still under investigation.

39. Successful expression of heterologous proteins in Paenibacillus larvae. L.

Poppinga, E. Genersch (Lnderinstitut fr Bienenkunde Hohen Neuendorf, Friedrich-Engels-Str. 32, 16540 Hohen Neuendorf, Germany)

The Gram-positive bacterium Paenibacillus larvae the causative agent of American Foulbrood (AFB) of honey bees, shows differences in virulence among the four genotypes ERIC I-IV. The two differently virulent P. larvae genotypes ERIC I and ERIC II are predominantly responsible for AFB outbreaks in Europe, with ERIC II being the more virulent strain on larval level. Recent comparative genomic and proteomic approaches revealed an S-layer protein (SplA) present in ERIC II but not in ERIC I strains, and which could act as a putative virulence factor during infection. In order to understand the roles of genotype specific putative virulence factors, molecular tools for genetic manipulation of this bacterium are urgently required and havent been explored so far. We therefore established a protocol for sucessfull transformation of the Gram-positive-E. coli shuttle plasmid pAD43-25 (BGSC), carrying the green fluorescent protein (GFP) mutant gfpmut3a under the control of the constitutive Bacillus cereus UW85 promoter Pupp for both genotypes ERIC I and ERIC II. The heterologous expression of GFP in P. larvae provides the first molecular tool for this species and is a prerequisite for future studies on molecular pathogenesis of AFB. The plasmid is further used as a tool for expression of histidin-labelled ERIC II specific S-layer protein SplA-His6 in the naturally SplA lacking ERIC I wildtype strain. His-tagged SplA is visualized in ERIC I mutants in silico and in vivo by labelling the recombinant protein with a Penta-His Alexa Fluor 488 conjugate in vegetative cells. ERIC I mutants were visualised by fluorescence microscopy and can be investigated in future infection studies.

40. Functional characterization of secondary metabolites of Paenibacillus larvae. E. Garcia-Gonzalez1, S. Mller2, S. Gisder1, R. Borriss3, R. Sssmuth2, E. Genersch1 (1 Lnderinstitut fr Bienenkunde Hohen Neuendorf, Molekulare Mikrobiologie und Bienenkrankheiten, 16540 Hohen Neuendorf, Germany; 2 Institut fr Chemie, Technische Universitt, Franklinstr. 28/29, 10587 Berlin, Germany)

American foulbrood (AFB) is considered the most contagious and destructive infectious disease in honeybees, caused by the Gram-positive, spore-forming bacterium Paenibacillus larvae. Recently, comparative genome analysis revealed that P. larvae harbours giant gene clusters that code for polyketide synthases (PKS) and nonribosomal

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peptide synthetases (NRPS). These enzymatic complexes are responsible for the biosynthesis of natural products that are implicated in multiple functions such as antibiotic, immunosuppressive, cytostatic and toxic activity. We hypothesized that P. larvae requires these substances in order to successfully compete against bacterial competitors present in the larvae, i.e. that the substances produced by these NRPS serve as antibiotics. On the other hand some PKS-related compounds have been proven to be highly toxic and were associated with virulence. In P. larvae genome, we so far identified, extended, and assembled three putative NRPS/PKS clusters. Using bacterial growth inhibition assays we were able to demonstrate that P. larvae strains indeed inhibited the growth of different bacterial species suggesting that at least some of these NRPS/PKS clusters are functional and lead to non-ribosomal antibiotic production. A knock-out mutant for one of these clusters revealed an alteration in antimicrobial substances production pattern. NRPS-Cluster II knock-out showed a dramatic decrease in bacterial growth inhibition. Further experiments, both in vitro and in vivo together with chemical characterization (HPLC-ESI-MS), are performed to prove the role of these substances in pathogenesis.

41. Cell culture models for DWV. N. Mckel, E. Genersch (Lnderinstitut fr

Bienenkunde Hohen Neuendorf, Friedrich-Engels-Str. 32, 16540 Hohen Neuendorf, Germany)

Deformed wing virus (DWV) is a viral pathogen of the European honeybee (Apis mellifera). To analyze the interaction between DWV and its target cells we recently established an in vitro-cell culture model for DWV using primary neuronal cells of honeybee pupae isolated from mushroom bodies. We also developed a cell culture from midgut cells from pupae, since the midgut is discussed as main attack area for DWV. However, cell culture models based on primary cells isolated from pupae have several disadvantages like lack of reproducibility, limited (i.e. seasonal) availability and the struggling to find or generate DWV free colonies, because this is a prerequisite for cell infection assays and analyzing DWV. We recently published the establishment of a cell culture model for honey bee pathogenic Nosema spp. based on the permanent cell line IPL-LD65Y, derived from the gypsy moth (Lymantria dispar). Encouraged through this success, we now aimed at finding a lepidopteran cell line permissive for DWV in order to establish a heterologous cell culture model for DWV. Several lepidopteran cell lines proved to be susceptible to DWV infection although replication efficiency varied between the cell lines. Successful infection with DWV could be demonstrated by DWV-specific RT-PCR. Viral replication could be demonstrated by strand-specific RT-PCR. These cell culture models provide a novel means to analyze DWV-host cell interactions and to develop new treatments against this pathogen.

42. Efficacy of the varroa treatment with half of the recommended doses of

rotenone in stripes in Rijeka, Croatia. M.