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1、 Proceedings of the COLOSS Work shop “Method standardization for larval tests” Graz, Austria, 7.-9.6.2010 FA 0803 COLOSS Workshop “Method standardization for larval tests”, 7.-9.6.2010, Graz, Austria 2 Dear colleagues, On behalf of the local organising team, it is my personal pleasure to welcome you
2、 to the Work shop “Method standardization for larval tests” in Graz, Austria. I would like to thank all the people who have helped to organise and conduct this meeting and of course all contributors for submitting their abstracts, which I hope will stimulate rewarding discussions on the method stand
3、ardization, trouble shooting and research conducted. The method to rear larvae will further advance honeybee science and enable us to answer several questions that cannot be solved inside a colony due to the complex system of rearing and cannibalism inside a colony. Financial support is granted by C
4、OST via the Action FA0803 COLOSS and the Karl- Franzens-University Graz. I am looking forward meeting all of you, and hope you will enjoy this work shop in Graz. Karl Crailsheim Vice Chair of COLOSS Graz, Austria, Tuesday, 25 May 2010 The local organising committee for the work shop and editors of t
5、his proceedings: Department of Zoology, Karl-Franzens-University Graz Robert Brodschneider, Karl Crailsheim, Ulrike Riessberger-Gall, Jutta Vollmann COLOSS Workshop “Method standardization for larval tests”, 7.-9.6.2010, Graz, Austria 3 Table of contents 2 Welcome 3 Table of contents 4 Program 5 Abs
6、tracts 5 Aupinel et al.: A new larval in vitro rearing method to test effects of pesticides on honey bee brood. 6 Bevk Medrzycki, P.; Fortini, D.; Michaud, B.; Tasei, J.N.; Odoux, J.F. A new larval in vitro rearing method to test effects of pesticides on honey bee brood. Redia. 2007, 90: 91-94. Aupi
7、nel, P.; Fortini, D.; Michaud, B.; Marolleau, F.; Tasei, J.N.; Odoux, J.F. Toxicity of dimethoate and fenoxycarb to honey bee brood (Apis mellifera), using a new in vitro standardized feeding method. Pest Management Science. 2007, 63 (11): 1090-1094. Aupinel, P.; Fortini, D.; Dufour, H.; Tasei, J.N.
8、; Michaud, B.; Odoux, J.F.; Pham-Delgue, M.H. Improvement of artificial feeding in a standard in vitro method for rearing Apis mellifera larvae. Bulletin of Insectology. 2005, 58 (2): 107-111. COLOSS Workshop “Method standardization for larval tests”, 7.-9.6.2010, Graz, Austria 6 How stress during l
9、arval development affects immature and adult honey bees? Danilo Bevk, Jasna Kralj National Institute of Biology, Vecna pot 111, 1000 Ljubljana, Slovenia Author for correspondence: Danilo.bevknib.si, Phone: +386 1 4233388 Bees are exposed to various stress factors during development such as diseases,
10、 pesticides and afflicted weather conditions. Development of honey bees could be thus affected directly by pathogens and other factors or indirectly by food. Impact of different stress factors in the period of bee development can be successfully tested by an in vivo method of rearing bee larvae. Bee
11、s are reared artificially in controlled environment by feeding larvae with known composition and amount of food. The method enables to test effects of stress factors on weight, larval development, survival and performance of adult bees. The method of artificial rearing of larvae demonstrated in work
12、shop in Graz will be applied at the National institute of biology in Slovenia to test influence of pathogens and sub-lethal doses of pesticides applied during larval development on longevity, flight activity, orientation and learning of adult bees. COLOSS Workshop “Method standardization for larval
13、tests”, 7.-9.6.2010, Graz, Austria 7 Effect of insect resistant (Bt) GM corn pollen on the development of honeybee (Apis mellifera) larvae in vitro. Lszl Bksi, Enik Szalai Mtray, Edit Zajcz, Lvia Harka Research Institute for Animal Breeding and Nutrition Research Group for Honeybee Breeding and Biol
14、ogy, H-2101 Gdll, Hungary Author for correspondence: bekesikatki.hu There is no officially adopted method for rearing honeybee larvae in vitro. The method recommended by the 91/EEC directive for testing bee larvae for toxic compounds, hides several errors (Aupinel et al., 2005). Publications of Remb
15、old and Lackner (1981); Brodsgaard et al. (1998); Malone et al. (2002) have been criticized recently, because of high mortality rate along rearing. In our experiments larvae of 3 days were transferred into U-bottom plastic cell culture plates. Our basic larval diet (BLD) contained d-glucose, d-fruct
16、ose, yeast extract and gentamycin. It was mixed with 1:1 fresh royal jelly. This feed was given 20 % pollen (1:1 beebread and Bt (MON810)- or isogenic corn pollen. The plates were incubated at 35 C with a relative moisture of 90 %. Beebread with royal jelly and pure royal jelly served as control fee
17、ds. The larvae were fed and weighed one by one two times a day. According to the results of our experiment the difference in weight between Bt- and iso groups was 18 % which may demonstrate the development breaking effect of Bt-pollen. After 72 hours larval mortality suddenly increased and only the
18、5-10 % of the larvae survived longer. In histological preparations of the midgut, the peritrophic membrane of larvae fed with Bt- pollen, seemed to be discontinuous and incomplete that might explain deficient digestion and lower weight gain. COLOSS Workshop “Method standardization for larval tests”,
19、 7.-9.6.2010, Graz, Austria 8 Quality of artificially reared honey bees. Robert Brodschneider, Ulrike Riessberger-Gall, Jutta Vollmann, Karl Crailsheim Department of Zoology, Karl-Franzens-University Graz, Universittsplatz 2, 8010 Graz, Austria Author for correspondence: robert.brodschneideruni-graz
20、.at To evaluate the quality of semi-defined diets for honey bee larvae we compared physiological parameters of adult bees reared according to the protocol of Aupinel et al. (2005) to sister bees reared under natural conditions in the colony. Artificially reared honey bee larvae gain sufficient nutri
21、ents to develop into adults capable of long, persisting flights. We demonstrated differences between artficially and naturally reared honey bees in top performance flight, wing size and dry weight of thorax in one experiment (Brodschneider et al., 2009a) and in 16 out of 18 or 17 out of 17, respecti
22、vely investigated parameters of body weight or size in another experiment including two colonies (Brodschneider et al., 2009b). Artificially reared larvae were always slightly inferiour compared to their naturally reared sisters, suggesting deficiencies in the larval diet or the way it is applied. T
23、he method for artificial larval rearing, though not yet being a totally chemically defined synthetic diet, enables research on nutritional requirements and malnourishment of developing larvae to be conducted in vitro. This method could be used in experiments helping to understand the effects of subl
24、ethal protein malnutrition during larval development as it may occur during shortage of pollen. Aupinel P., Fortini D., Dufour H., Tasei J.N., Michaud B., Odoux J.F., Pham-Delgue M.H. (2005) Improvement of artificial feeding in a standard in vitro method for rearing Apis mellifera larvae, Bull. Inse
25、ct 58, 107111. Brodschneider R., Riessberger-Gall U., Crailsheim K. (2009a) Flight performance of artificially reared honeybees (Apis mellifera), Apidologie 40, 441449. Brodschneider R., Steiner D., Moder A., Vollmann J., Riessberger-Gall U., Crailsheim K. (2009b) Synthetic larval diet produces ligh
26、ter and smaller honeybees (Apis mellifera), Apidologie 40, 663664. COLOSS Workshop “Method standardization for larval tests”, 7.-9.6.2010, Graz, Austria 9 Virulence of different Melissococcus plutonius strains tested by an in vitro larval test. J.D. Charrire; V. Kilchenmann, A. Roetschi Swiss Bee Re
27、search Centre, Agroscope Liebefeld-Posieux Research Station ALP, CH-3003 Bern, Switzerland Author for correspondence: jean-daniel.charrierealp.admin.ch In Switzerland, European foulbrood (EFB) is a honeybee disease which requires control in accordance with the Swiss Animal Epidemic Regulation. After
28、 having been under control for the last 30 years, cases have recently been reported with increased frequency. Between 1970 and 1998, approximately 20 to 50 diseased apiaries per year were sanitized by the veterinary authorities. However, since 1999 there has been a significant increase of reported c
29、ases, with more than 790 apiaries affected in 2009 alone. This represents a prevalence of 4.2 %. At the moment, we have no explanation for the dramatic expansion of this highly infectious brood disease. Significantly, we also observe that currently several regions in Switzerland are free of EFB as i
30、n most of Europe where EFB reports are anecdotal and present with low morbidity. An exception being the UK where the prevalence of EFB is also rather high. One hypothesis to explain the resurgence of EFB in Switzerland and the other isolated regions where EFB is problematic could be the emergence of
31、 a more virulent Melissococcus plutonius strain. To test this hypothesis, we used the larval in vitro rearing method developed by Aupinel et al. (Pest. Manag. Sci., 2007) with minor modifications. Here, we compare different Swiss, French and Italian EFB strains and present the first results of these
32、 tests COLOSS Workshop “Method standardization for larval tests”, 7.-9.6.2010, Graz, Austria 10 Evaluation of the effects from probiotic bacteria on Paenibacillus larvae using in vitro rearing of honeybee larvae. Eva Forsgren Department of Ecology, Swedish University of Agricultural Sciences Author
33、for correspondence: Eva.Forsgrenekol.slu.se Exposure bioassays (in vitro rearing of honeybee larvae) were used to evaluate the antagonistic effects of newly identified lactic acid bacteria (LAB) originating from the honey stomach on the honeybee pathogen, Paenibacillus larvae. To rear larvae in vitr
34、o a protocol by Aupinel et al. (2005) was followed with minor modifications. Worker honey bee larvae were reared in 48-well tissue culture plates on a diet consisting of 50% royal jelly (v/v), and 50% of an aqueous solution of D-glucose (12%) and D-fructose (12%). Before any experiment, the required
35、 amount of diet was prepared and then stored at +4 for the duration of the experimental feeding. Before grafting, each plastic well was supplied with the pre- warmed diet. The control group was provided with uninfected diet while the experiment groups were either provided larval diet spiked with kno
36、wn amounts of P. larvae spores or P. larvae spores mixed with the LAB mixture. First instar worker larvae (less than 24 hours) were grafted from larval combs and transferred to the surface of the larval diet of the different treatments. The larvae were kept in an incubator at 35 C until the experime
37、nt was finished 14 days post-infection. Throughout the experiment dead larvae were removed daily and cultured on agar plates to verify presence or absence of P. larvae. Adding the LAB mixture to the larval food significantly reduced the number of AFB infected larvae in the exposure bioassays. The re
38、sults demonstrate that honey bee specific LAB possess beneficial properties for honeybee health. The use of exposure bioassays as well as possible benefits from enhancing growth of LAB or from applying LAB to honeybee colonies will be discussed. COLOSS Workshop “Method standardization for larval tes
39、ts”, 7.-9.6.2010, Graz, Austria 11 Experiences with artificial feeding in a standardized in vitro method for rearing larvae of the honeybee Apis mellifera. Nicole Hanewald BASF SE, APD/EE - LI425, 67117 Limburgerhof, Germany Author for correspondence: , Phone: +49 621 60-27189, Fax: +49 621 60-27214
40、, BASF works on the standardization of the method proposed by Aupinel since 2007 (member of the ring test group). After establishing the method and reaching acceptable control mortalities we started to work on simplifications and improvements to the method of Aupinel. To give a few examples: we used
41、 one instead of three diets, reduced the caging time of the queens, reared in different climatic chambers and incubators, etc. LR50 and NOECs were determined for Dimethoate and Fenoxycarb as proposed by the ring test group led by P. Aupinel. As observations during the last years showed further resea
42、rch should be done concerning: healthiness of the colonies during the course of a year (increasing control mortalities starting end of July), stage of the larvae and diseases quality of the Royal Jelly the need of three different kind of diet caging time of the queens climatic conditions during the
43、conduct of the study comparison acute and chronic feeding BASF would be very interested in a comparison of results derived from studies done in the laboratory and results from semi-field and field trails to have a kind of validation and an evidence of the sensitivity of the method. COLOSS Workshop “
44、Method standardization for larval tests”, 7.-9.6.2010, Graz, Austria 12 Artificial larval rearing as a tool for evaluating the effects of imidacloprid on honey bees under laboratory conditions. Fani Hatjina, Leonidas Charistos Hellenic Institute of Apiculture, N. Moudania, 63 200, Greece Author for
45、correspondence: fhatjinainstmelissocomias.gr, Phone: +302373091297 A current project in Hellenic Institute of Apiculture concerns the evaluation of the effects of the neonicotinoid imidacloprid on honey bees using in-vivo and in-vitro methodology. One of the in-vitro methods already used in our Inst
46、itute is the use of hoarding cages where the effect of imidacloprid was evaluated on adult honey bee longevity and development of HPGs. However, artificial larval rearing is increasingly being used as an in-vitro method for assessing the toxicity of different substances to developing stages of honey
47、 bees. Imidacloprid may be very dangerous to the larvae, and cause damages at the colony level later on, even if apparently not harmful to individual adult bees. The first test on artificial larval rearing will be run in our laboratory during April-May 2010 using 96-well tissue culture plates accord
48、ing to Aupinel et al. 2005 and Aupinel et al. 2007. Our aim is to define and standardize the methodology in accordance with other laboratories in order to have undisputable results on risk assessment of plant protection products on honey bees and to discuss other possible applications of the methodo
49、logy. COLOSS Workshop “Method standardization for larval tests”, 7.-9.6.2010, Graz, Austria 13 Artificial rearing of honey bee larvae to investigative economically important diseases in South Africa. Hannelie Human SIRG, Dept Zoology and Entomology, University of Pretoria, Pretoria 0002 Author for correspondence: hhumanzoology.up.ac.za There are a number of reasons for rearing worker bee larvae (Apis mellifera) under laboratory conditions, such as the effects of antibiotics and toxicity of insecticides