Comparison of two methods to assess effects of insecticides on hypopharyngeal gland development of honey bee

contribution to session IV Test methodolog

Sublethal effects of fipronil on the ability of honeybees (Apis mellifera L.) to orientate in a complex maze

contribution to session IVTest methodology Background: The recent fipronil-based pesticide is accused by bee-keepers of causing depopulations in hives of honeybees (Apis mellifera L.). Behavioural effects during the flight of foraging honeybees would have been evoked. To test whether the insecticide fipronil may disorientate foragers, its impact on orientation in a maze was examined. Bees had to fly through a sequence of boxes to reach the target, which was a feeder containing a reward of sugar solution. After being trained to associate a green mark with the reward, foragers received 1 μg kg-1 fipronil orally and their capacity to orientate through the maze following the colour mark was tested and compared to control. Results: The rate of foragers entering the maze, and so responding to the mark placed at the entrance, was reduced with fipronil-fed animals. Before and after treatment, 86-89% of bees equally flew through the whole path and arrived to the goal without mistakes. The rate of fipronil-treated bees finding path without mistakes decreased to 60%. Conversely, the rate of bees with unsuccessful searches for the goal notably increased with treatment (34% in treated bees versus 4% in control bees). Conclusion: Our results show that orientation capacities of foragers in a complex maze were affected by fipronil. Keywords: Apis mellifera L., pesticide, maze, conditioning, visual learning, flight

The homing flight method to assess the effect of sublethal doses of plant protection products on the honey bee in field conditions: results of the ring tests

The evaluation of the potential effects of plants protection products on honeybee behavior is considered as part of the risk assessment according to Regulation (EC) No 1107/2009 and the EFSA Guidance document (EFSA 2013). But no standardized and validated method is still available. With current revisions of plant protection product risk assessment on the honeybee, a European ring test is conducted since 2015 with 11 voluntary laboratories to test a methodology assessing the effects of sublethal doses of a plant protection product administered in controlled conditions on the homing capacity of forager bees in the field. Homing success is measured by monitoring free-ranging honey bees with radio-frequency identification (RFID) tagging technology

3.5 The homing flight ring test: method for the assessment of sublethal doses of plant protection products on the honey bee in field conditions

International audienc

Honey bee brood ring-test: method for testing pesticide toxicity on honeybee brood in laboratory conditions

contribution to session IV Test methodology The Experimental unit of entomology (INRA, France) developed a new in vitro method to assess effects of pesticides on honey bee larvae. The method consists in rearing bee larvae in plastic cells. The larvae are fed with diet containing 50% of fresh royal jelly and 50% of an aqueous sugar and yeast extract solution, and reared in an incubator at 35 °C and 96% relative humidity. According to that method, 9 tests (7 in 2008 and 2 in 2005) were carried out in 7 laboratories and different countries. The objective of these trials was to assess the LD50 for dimethoate 48 hours after an acute exposure. The LD50 values ranged from 1.5 μg a.i./larva to 8.8 μg a.i./larva, with 2 tests with particularly high values (5.0 and 8.8 μg a.i./larva). In 7 tests, these values ranged from 1.5 μg a.i./larva to 3.1 μg a.i./larva. Such variability may be due to the colony origin, the season and larva heterogeneity at grafting. Solutions are proposed to improve the method through the continuation of the ring test. Keywords: Apis mellifera, brood, in vitro test, dimethoat

Predictive systems models can help elucidate bee declines driven by multiple combined stressors

Published onlineThis is the author accepted manuscript. The final version is available from Springer Verlag via the DOI in this record.Bee declines are driven by multiple combined stresses, making it exceedingly difficult to identify experimentally the most critical threats to bees and their pollination services. We highlight here the too often ignored potential of mechanistic models in identifying critical stress combinations. Advanced bee models are now available as open access tools and offer an unprecedented opportunity for bee biologists to explore bee resilience tipping points in a variety of environmental contexts. We provide general guidelines on how to run bee models to help detect a priori critical stress combinations to be targeted in the field. This so-called funnel analysis should be performed in tight conjunction with the recent development of large-scale field monitoring programs for bee health surveillance.This work was supported by the European Community program (797/2004) for French beekeeping coordinated by the French Ministry of Agriculture (RISQAPI project). JLO, MAB, and PK were funded by grants from the Biotechnology and Biological Sciences Research Council, UK [BB/J014915/1; BB/K014463/1]. We thank Axel Decourtye and Jean-François Odoux for useful discussions on honeybee colony modeling and field monitoring design, as well as two anonymous reviewers for constructive comments on the manuscript

The distribution of Aspergillus spp. opportunistic parasites in hives and their pathogenicity to honey bees

Stonebrood is a disease of honey bee larvae caused by fungi from the genus Aspergillus. As very few studies have focused on the epidemiological aspects of stonebrood and diseased brood may be rapidly discarded by worker bees, it is possible that a high number of cases go undetected. Aspergillus spp. fungi are ubiquitous and associated with disease in many insects, plants, animals and man. They are regarded as opportunistic pathogens that require immunocompromised hosts to establish infection. Microbiological studies have shown high prevalences of Aspergillus spp. in apiaries which occur saprophytically on hive substrates. However, the specific conditions required for pathogenicity to develop remain unknown. In this study, an apiary was screened to determine the prevalence and diversity of Aspergillus spp. fungi. A series of dose–response tests were then conducted using laboratory reared larvae to determine the pathogenicity and virulence of frequently occurring isolates. The susceptibility of adult worker bees to Aspergillus flavus was also tested. Three isolates (A. flavus, Aspergillus nomius and Aspergillus phoenicis) of the ten species identified were pathogenic to honey bee larvae. Moreover, adult honey bees were also confirmed to be highly susceptible to A. flavus infection when they ingested conidia. Neither of the two Aspergillus fumigatus strains used in dose–response tests induced mortality in larvae and were the least pathogenic of the isolates tested. These results confirm the ubiquity of Aspergillus spp. in the apiary environment and highlight their potential to infect both larvae and adult bees

Diversity of Melissococcus plutonius from Honeybee Larvae in Japan and Experimental Reproduction of European Foulbrood with Cultured Atypical Isolates

European foulbrood (EFB) is an important infectious disease of honeybee larvae, but its pathogenic mechanisms are still poorly understood. The causative agent, Melissococcus plutonius, is a fastidious organism, and microaerophilic to anaerobic conditions and the addition of potassium phosphate to culture media are required for growth. Although M. plutonius is believed to be remarkably homologous, in addition to M. plutonius isolates with typical cultural characteristics, M. plutonius-like organisms, with characteristics seemingly different from those of typical M. plutonius, have often been isolated from diseased larvae with clinical signs of EFB in Japan. Cultural and biochemical characterization of 14 M. plutonius and 19 M. plutonius-like strain/isolates revealed that, unlike typical M. plutonius strain/isolates, M. plutonius-like isolates were not fastidious, and the addition of potassium phosphate was not required for normal growth. Moreover, only M. plutonius-like isolates, but not typical M. plutonius strain/isolates, grew anaerobically on sodium phosphate-supplemented medium and aerobically on some potassium salt-supplemented media, were positive for β-glucosidase activity, hydrolyzed esculin, and produced acid from L-arabinose, D-cellobiose, and salicin. Despite the phenotypic differences, 16S rRNA gene sequence analysis and DNA-DNA hybridization demonstrated that M. plutonius-like organisms were taxonomically identical to M. plutonius. However, by pulsed-field gel electrophoresis analysis, these typical and atypical (M. plutonius-like) isolates were separately grouped into two genetically distinct clusters. Although M. plutonius is known to lose virulence quickly when cultured artificially, experimental infection of representative isolates showed that atypical M. plutonius maintained the ability to cause EFB in honeybee larvae even after cultured in vitro in laboratory media. Because the rapid decrease of virulence in cultured M. plutonius was a major impediment to elucidation of the pathogenesis of EFB, atypical M. plutonius discovered in this study will be a breakthrough in EFB research