Standard methods for chemical ecology research in Apis mellifera.

Authors: Baldwyn Torto, Mark J Carroll, Adrian Duehl, Ayuka T Fombong, Tamar Katzav Gozansky, Francesco Nazzi, Victoria Soroker, Peter E A Teal.

Table of contents

Authors
Summary
1. Introduction
2. In situ volatile collection of odours in the colony environment
   2.1. Introduction
   2.2. Collection and analyses of honey bee volatiles

      2.2.1. Volatiles in the headspace environment
         2.2.1.1. Volatile sampling in the headspace environment
      2.2.2. Collection and recovery (desorption) of volatiles
         2.2.2.1. Solvent desorption of volatiles
         2.2.2.2. Thermal desorption of volatiles
            2.2.2.2.1. Thermal desorption of static headspace volatiles by SPME
            2.2.2.2.2. Thermal desorption of dynamic headspace volatiles by Tenax
         2.2.2.3. Sampling odours at the whole colony scale
         2.2.2.4. Sampling odours at a whole frame scale
      2.2.3. Separation and analysis of volatiles by gas chromatography (GC)
         2.2.3.1. Injector
         2.2.3.2. Column
         2.2.3.3. Run parameters:
            2.2.3.3.1. Column flow parameters
            2.2.3.3.2. Oven temperature ramp
            2.2.3.3.3. MS detector parameters
      2.2.4. Detection and analysis of volatiles
         2.2.4.1. Identification of volatile and non-volatile compounds
            2.2.4.1.1. Using GC-MS to identify sample peaks
            2.2.4.1.2. Quantification of volatile and non-volatile compounds with internal standards
   2.3. Conclusion
3. Ex-situ collection of honey bee odours and electrophysiology
   3.1. Introduction
   3.2. Volatile collection

      3.2.1. Setup and volatile sampling
      3.2.2. Dynamic volatile collection
      3.2.3. Static volatile collection
   3.3. Electrophysiology
      3.3.1. Setup
      3.3.2. Types of electrodes
         3.3.2.1. Glass capillary electrodes
         3.3.2.2. Probe electrodes
      3.3.3. Sensory organ preparation and mounting
      3.3.4. EAG recording
      3.3.5. Coupled gas chromatography-electroantennographic detection (GC-EAD) recording
      3.3.6. Discontinuous EAG recording
   3.4. Chemical identification of electrophysiologically-active components
   3.5. Summary
4. Extraction and analysis of honey bee non-volatile cuticular hydrocarbons
   4.1. Introduction
   4.2. Techniques for analysing honey bee CHC

      4.2.1. Extraction
      4.2.2. Sample preparation
      4.2.3. Identification
         4.2.3.1. Analysis
         4.2.3.2. Double bond position in unsaturated hydrocarbons
         4.2.3.3. Stereochemistry of alkenes (determining the identities of geometric isomers of    unsaturated hydrocarbons)
         4.2.3.4. Branching position
         4.2.3.5. Synthesis for the purpose of identification     
      4.2.4. Data analysis
5. Bee attraction bioassay
   5.1. Introduction
   5.2. Stimuli preparation

      5.2.1. Preparation of synthetic esters
   5.3. Stimuli presentation techniques
      5.3.1. Use of surrogates
      5.3.2. Stimuli preparation
   5.4. Bioassays:       
      5.4.1. In colony assays
      5.4.2. Choice assays on groups in semi natural conditions (micro-hives)
      5.4.3. Arena tests
         5.4.3.1 An example of arena choice bioassay using live workers
   5.5. Summary
6. In vitro bioassay for studying mechanisms regulating pheromonal gland activity in honey bees
   6.1. Introduction
   6.2. Methods

      6.2.1. Isolation of organs/tissues
      6.2.2. Maintaining the normal performance of the gland in an artificial medium
      6.2.3. Media contamination by microorganisms
      6.2.4. Selection and labelling of an appropriate precursor
      6.2.5. Incubation conditions
      6.2.6. Determining optimum incubation time.
      6.2.7. Extraction of pheromone.
      6.2.8. Product analysis.
         6.2.8.1. Dissection and sample preparation
         6.2.8.2 Bee incubation medium preparation based on Kaatz (1985) amino acids (AA) (50 ml):
         6.2.8.3. Isolation and identification of Dufour's biosynthesis products.
   6.3. Summary
7. Acknowledgements
8. References