Miscellaneous standard methods for Apis mellifera research.

Authors: Hannelie Human, Robert Brodschneider, Vincent Dietemann, Galen Dively, James D Ellis, Eva Forsgren, Ingemar Fries, Fani Hatjina, Fu-Liang Hu, Rodolfo Jaffé, Annette Bruun Jensen, Angela Köhler, Joseph Magyar, Asli Özikrim, Christian W W Pirk, Robyn Rose, Ursula Strauss, Gina Tanner, David R Tarpy, Jozef J M van der Steen, Anthony Vaudo, Fleming Vejsnæs, Jerzy Wilde, Geoffrey R Williams, Huo-Qing Zheng.

Table of contents

1. Introduction
2. Research methods at the individual level
   2.1. Standard methods for immobilising, killing and storing adult Apis mellifera in the laboratory

      2.1.1. Introduction
      2.1.2. Immobilising adults Physical immobilisation Chemical and physical immobilisation
   Carbon dioxide
   Anaesthesia considerations
      2.1.3. Killing adults Thermal killing
   Heat Mechanical killing Chemical killing
      2.1.4. Storing dead adults
   2.2. Determination of individual bee weight
      2.2.1. Balance required for weighing individual bees or larvae or body parts
      2.2.2. Weighing of larvae
      2.2.3. Weighing of adult honey bees
      2.2.4. Weighing body parts
      2.2.5. Determining dry weight
   2.3. Microinjection
      2.3.1. Introduction
      2.3.2. Microinjection using a Hamilton syringe
      2.3.3. Microinjection of small volumes using the Nanoject device and other micro injectors
      2.3.4. Perspectives
   2.4. Marking honey bee queens
      2.4.1. Colour-marking queens Marking type Procedure for paint marking Procedure for marking with Opalith discs Colour-marking code
      2.4.2. Clipping queens’ wings
   2.5. Obtaining brood and adults of known age
      2.5.1. Obtaining brood of known age Procedure to obtain worker or drone brood of known age Procedure to obtain queen brood of known age
      2.5.2. Obtaining pupae of known age
      2.5.3. Recognising the instar of larvae
      2.5.4. Recognising the age of larvae
      2.5.5. Recognising the age of pupae
      2.5.6. Obtaining workers of known age
      2.5.7. Conclusion
3. Other equipment used in the laboratory
   3.1. Using a haemocytometer to estimate the concentration of cells, spores or sperms

      3.1.1. Total or microscopic count
4. Research methods at the colony level
   4.1. Weighing full hives

      4.1.1. Introduction
      4.1.2. The Capaz hive scale
      4.1.3. The honey meter
      4.1.4. The use of the data from an electronic scale
   4.2. Using beelines to locate wild honey bee colonies
      4.2.1. Introduction
      4.2.2. Suggested materials
      4.2.3. Establishing a beeline Setting up a feeding station
      4.2.4. Following the beeline Observing beelines Tracking the beeline Using a mobile feeding station
      4.2.5. Locating the honey bee nest
      4.2.6. Alternative methods Following bees from water sources Beelining with a bee box Triangulating with feeding stations Calculating the distance between a honey bee nest and feeding station by timing a forager’s round trip
   4.3. Honey bee colony density estimations
      4.3.1. Determining a colony density index using feeding stations Material used Procedure Index data Statistical analyses
      4.3.2. Determination of honey bee colony density using genetic markers
      4.3.3. Sampling Drone sampling Worker sampling Genotyping Genetic diversity measures and reconstruction of queen genotypes Non-detection and non-sampling errors Density estimation
      4.3.4. Future research needs and perspectives
   4.4. Estimating the number of dead honey bees expelled from a honey bee colony with a trap
      4.4.1 Aim of using dead bee traps
      4.4.2. Limitations of using dead bee traps
      4.4.3. Types of dead bee traps
      4.4.4. Dead bee traps requirements as gathered from the literature
      4.4.5. Recommended dead bee traps to use
      4.4.6. Building a dead bee trap
      4.4.7. Protocol for calibrating dead bees in traps
      4.4.8. Protocol for using a dead bee trap
      4.4.9. Dead bee trap trade-offs
   4.5. Creating multiple queen colonies
      4.5.1. Mandible clipping procedure
      4.5.2. Preparation of colonies destined to host the multiple queens
      4.5.3. Steps for maintenance of an artificially established multiple-queen social organisation
   4.6. Digital monitoring of brood development via location recognition
      4.6.1. Introduction
      4.6.2. Procedure for data acquisition Software requirements Before starting the project Image acquisition.
      4.6.3. Image analysis Analysis of the first image (BFD 00) For all consecutive images (BFD + 05, 10, 16, 22)
      4.6.4. Finalization of the analysis.
      4.6.5. Conclusion
   4.7. Collecting pollen and nectar from bees and flowers
      4.7.1 Introduction
      4.7.2. Methods for pollen collection Collection of fresh pollen from flowers
   Using paper bags to collect fresh pollen
   Manual collection of fresh pollen
   Using a paint brush for collection of fresh pollen
   Collection of fresh pollen from smaller flowers such as canola. Collection of bee collected pollen using pollen traps Ensuring quality of bee collected pollen
      4.7.3. Nectar collection Collecting nectar from honey bees Nectar collection from flowers
      4.7.4. Precautions when sampling pollen and nectar for residue analyses
5. Acknowledgements
6. References