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Standard methods for chemical ecology research in Apis mellifera

Summary:

This paper describes basic methods essential in elucidating chemically-mediated behavioural interactions among honey bees, and between honey bees and other arthropods. These range from bioassay methods used to demonstrate the role of specific behaviours, techniques and equipment used to collect and analyse semiochemicals (both volatiles and non-volatiles e.g. cuticular hydrocarbons) from individual honey bees, groups of bees or an entire colony in its native environments. This paper covers: collection and analysis of honey bee volatiles in the natural environment, collection and analysis of bee volatiles out of their natural environment and their antennal detection, collection and analysis of non-volatile cuticular hydrocarbons, bioassays with queen pheromone and finally a section focusing on in vitro bioassays as a tool for elucidation of mechanisms regulating pheromone gland activity.

    1. 2.1. Introduction
    2. 2.2. Collection and analyses of honey bee volatiles
    3. 2.2.1. Volatiles in the headspace environment
    4. 2.2.1.1. Volatile sampling in the headspace environment
    5. 2.2.2. Collection and recovery (desorption) of volatiles
    6. 2.2.2.1. Solvent desorption of volatiles
    7. 2.2.2.2. Thermal desorption of volatiles
    8. 2.2.2.2.1. Thermal desorption of static headspace volatiles by SPME
    9. 2.2.2.2.2. Thermal desorption of dynamic headspace volatiles by Tenax
    10. 2.2.2.3. Sampling odours at the whole colony scale
    11. 2.2.2.4. Sampling odours at a whole frame scale
    12. 2.2.3. Separation and analysis of volatiles by gas chromatography (GC)
    13. 2.2.3.1. Injector
    14. 2.2.3.2. Column
    15. 2.2.3.3. Run parameters
    16. 2.2.3.3.1. Column flow parameters
    17. 2.2.3.3.2. Oven temperature ramp
    18. 2.2.3.3.3. MS detector parameters
    19. 2.2.4. Detection and analysis of volatiles
    20. 2.2.4.1. Identification of volatile and non-volatile compounds
    21. 2.2.4.1.1. Using GC-MS to identify sample peaks
    22. 2.2.4.1.2. Quantification of volatile and non-volatile compounds with internal standards
    23. 2.3. Conclusion
    1. 3.1. Introduction
    2. 3.2. Volatile collection
    3. 3.2.1. Setup and volatile sampling
    4. 3.2.2. Dynamic volatile collection
    5. 3.2.3. Static volatile collection
    6. 3.3. Electrophysiology
    7. 3.3.1. Setup
    8. 3.3.2. Types of electrodes
    9. 3.3.2.1. Glass capillary electrodes
    10. 3.3.2.2. Probe electrodes
    11. 3.3.3. Sensory organ preparation and mounting
    12. 3.3.4. EAG Recording
    13. 3.3.5. Coupled gas chromatographyelectroantennographic detection (GC-EAD) recording
    14. 3.3.6. Discontinuous EAG recording
    15. 3.4. Chemical identification of electrophysiologicallyactive components
    16. 3.5. Summary
    1. 4.1. Introduction
    2. 4.2. Techniques for analysing honey bee CHC
    3. 4.2.1. Extraction
    4. 4.2.2. Sample preparation
    5. 4.2.3. Identification
    6. 4.2.3.1. Analysis
    7. 4.2.3.2. Double bond position in unsaturated hydrocarbons
    8. 4.2.3.3. Stereochemistry of alkenes (Determining the identities of geometric isomers of unsaturated hydrocarbons)
    9. 4.2.3.4. Branching position
    10. 4.2.3.5. Synthesis for the purpose of identification
    11. 4.2.4. Data analysis
    1. 5.1. Introduction
    2. 5.2. Stimuli preparation
    3. 5.2.1. Preparation of synthetic esters
    4. 5.3. Stimuli presentation techniques
    5. 5.3.1. Use of surrogates
    6. 5.3.2. Stimuli preparation
    7. 5.4. Bioassays:
    8. 5.4.1. In colony assays
    9. 5.4.2. Choice assays on groups in semi natural conditions (micro-hives)
    10. 5.4.3. Arena tests
    11. 5.4.3.1. An example of arena choice bioassay using live workers
    12. 5.5. Summary
    1. 6.1. Introduction
    2. 6.2. Methods
    3. 6.2.1. Isolation of organs/tissues
    4. 6.2.2. Maintaining the normal performance of the gland in an artificial medium
    5. 6.2.3. Media contamination by microorganisms
    6. 6.2.4. Selection and labelling of an appropriate precursor
    7. 6.2.5. Incubation conditions
    8. 6.2.6. Determining optimum incubation time
    9. 6.2.7. Extraction of pheromone
    10. 6.2.8. Product analysis
    11. 6.2.8.1. Dissection and sample preparation
    12. 6.3. Summary
    13. 6.2.8.2. Bee incubation medium preparation based on Kaatz (1985)
    14. 6.2.8.3. Isolation and identification of Dufour’s biosynthesis products