Standard methods for maintaining adult Apis mellifera in cages under in vitro laboratory conditions.

Authors: Geoffrey R Williams, Cédric Alaux, Cecilia Costa, Támas Csáki, Vincent Doublet, Dorothea Eisenhardt, Ingemar Fries, Rolf Kuhn, Dino P McMahon, Piotr Medrzycki, Tomás E Murray, Myrsini E Natsopoulou, Peter Neumann, Randy Oliver, Robert J Paxton, Stephen F Pernal, Dave Shutler, Gina Tanner, Jozef J M van der Steen, and Robert Brodschneider.

Table of contents

Authors
Summary
1. General introduction
2. Experimental design

   2.1. Important experimental design considerations before caging adult workers in the laboratory
   2.2. Independence of observations for laboratory cage experiments involving adult workers
   2.3. Appropriate worker and cage replicates for laboratory experiments involving adult workers
   2.4. Appropriate randomization of study organisms for laboratory cage experiments involving adult workers
3. Statistical analyses
  
3.1. Where the response variable is not mortality during laboratory experiments involving adult workers
   3.2. Where the response variable is mortality during laboratory experiments involving adult workers
   3.3. Statistical software for laboratory experiments involving adult workers
4. Obtaining adult workers for laboratory experiments
   4.1. Considerations for choosing and obtaining adult workers for laboratory experiments

      4.1.1. Seasonal timing of adult worker collection for laboratory experiments
      4.1.2. Subspecies of adult workers used for laboratory experiments
      4.1.3. Age of adult workers used for laboratory experiments
      4.1.4. Queen status of source colonies used to obtain adult workers for laboratory experiments
      4.1.5. Strength of source colonies used to obtain adult workers for laboratory experiments
      4.1.6. Health of source colonies used to obtain adult workers for laboratory experiments
      4.1.7. Beekeeper management of source colonies used to obtain adult workers for laboratory experiments
      4.1.8. Environment surrounding source colonies used to obtain adult workers for laboratory experiments
      4.1.9. Weather before and during collection of adult workers for laboratory experiments
      4.1.10. Diurnal timing of collection of adult workers for laboratory experiments
   4.2. Collecting newly emerged workers for laboratory experiments
      4.2.1. Considerations for choosing to use newly emerged workers for laboratory experiments
      4.2.2. Obtaining newly emerged workers for laboratory experiments without caging queens
      4.2.3. Obtaining newly emerged workers for laboratory experiments by caging queens
      4.2.4. Obtaining newly emerged workers for laboratory experiments by in vitro rearing
   4.3. Collecting adult workers of an undefined age for laboratory experiments
      4.3.1. Considerations for choosing to use adult workers of an undefined age for laboratory experiments
      4.3.2. Challenges associated with collecting adult workers of an undefined age for laboratory experiments
      4.3.3. Collecting flying adult workers of an undefined age for laboratory experiments
         4.3.3.1. Collecting flying adult workers of an undefined age for laboratory experiments using a forceps
         4.3.3.2. Collecting flying adult workers of an undefined age for laboratory experiments using a container
         4.3.3.3. Collecting flying adult workers of an undefined age for laboratory experiments using an entrance trap
            4.3.3.3.1. Bologna Trap description for collecting adult workers for laboratory experiments
            4.3.3.3.2. Collecting flying adult workers of an undefined age for laboratory experiments using the Bologna Trap
      4.3.4. Collecting intra-hive adult workers of an undefined age for laboratory experiments
   4.4. Recommendations for choosing and collecting adult workers for laboratory experiments
5. Cages in which to maintain adult workers in the laboratory
   5.1. Types of cages in which to maintain adult workers in the laboratory
   5.2. Choosing a suitable cage to maintain adult workers in the laboratory

      5.2.1. Minimum criteria for frame and hoarding cages in which to maintain adult workers in the laboratory
      5.2.2. Supplementary frame and hoarding cage materials to be used when maintaining adult workers in the laboratory
      5.2.3. Minimum criteria for isolation cages in which to maintain adult workers in the laboratory
   5.3. Suitable cages in which to maintain adult workers in the laboratory
      5.3.1. Example of a frame cage in which to maintain adult workers in the laboratory
      5.3.2. Examples of hoarding cages in which to maintain adult workers in the laboratory
      5.3.3. Examples of isolation cages in which to maintain adult workers in the laboratory
6. Incubator conditions
   6.1. Regulation of biophysical properties within colonies
   6.2. Temperature

      6.2.1. Honey bee intra-hive temperature requirements
      6.2.2. Recommendations for incubator temperature for maintaining adult workers in the laboratory
   6.3. Relative humidity
      6.3.1. Honey bee intra-hive relative humidity requirements
      6.3.2. Regulating incubator relative humidity for maintaining adult workers in the laboratory
         6.3.2.1. Regulating incubator relative humidity for maintaining adult workers in the laboratory using an open water basin
         6.3.2.2. Regulating incubator relative humidity for maintaining adult workers in the laboratory using a saturated salt solution
            6.3.2.2.1. Criteria for using saturated salts to regulate incubator relative humidity for maintaining adult workers in the laboratory
            6.3.2.2.2. Choosing appropriate saturated salts for regulating incubator relative humidity for maintaining adult workers in the laboratory
            6.3.2.2.3. Preparing a saturated salt solution for regulating incubator relative humidity for maintaining adult workers in the laboratory
      6.3.3. Monitoring and recording incubator relative humidity when maintaining adult workers in the laboratory
      6.3.4. Recommendations for incubator relative humidity for maintaining adult workers in the laboratory
   6.4. Light
      6.4.1. Natural honey bee light conditions
      6.4.2. Recommendations for incubator light conditions for maintaining adult workers in the laboratory
   6.5. Ventilation
      6.5.1. Honey bee ventilation requirements
      6.5.2. Recommendations for incubator ventilation with ambient air for maintaining adult workers in the laboratory
7. Nutrition
   7.1. Nutritional requirements of worker honey bees
   7.2. Carbohydrates

      7.2.1. Types of carbohydrates to provide to caged adult workers in the laboratory
         7.2.1.1. Providing honey to caged adult workers in the laboratory
         7.2.1.2. Providing sucrose solution to caged adult workers in the laboratory
         7.2.1.3. Providing sucrose paste to caged adult workers in the laboratory
      7.2.2. Feeding devices for providing carbohydrates to caged adult workers in the laboratory
      7.2.3. Measuring carbohydrate consumption by caged adult workers in the laboratory
      7.2.4. Replenishing carbohydrates provided to caged adult workers in the laboratory
      7.2.5. Recommendations for providing carbohydrates to caged adult workers in the laboratory
   7.3. Proteins
      7.3.1. Types of proteins to provide to caged adult workers in the laboratory
         7.3.1.1. Providing bee bread to caged adult workers in the laboratory
         7.3.1.2. Providing corbicular pollen to caged adult workers in the laboratory
            7.3.1.2.1. Collecting and storing corbicular pollen to feed to caged adult workers in the laboratory
         7.3.1.3. Providing pollen substitutes to caged adult workers in the laboratory
      7.3.2. Feeding devices for providing proteins to caged adult workers in the laboratory
      7.3.3. Measuring protein consumption by caged adult workers in the laboratory
      7.3.4. Replenishing proteins provided to caged adult workers in the laboratory
      7.3.5. Recommendations for providing proteins to caged adult workers in the laboratory
   7.4. Lipids, minerals, and vitamins
   7.5. Water
   7.6. Food sterilisation and detoxification

      7.6.1. Pathogens and environmental contaminants found in bee products
      7.6.2. Sterilising bee products to destroy pathogens
         7.6.2.1. Sterilising bee products to destroy pathogens using radiation
         7.6.2.2. Sterilising bee products to destroy pathogens using temperature
      7.6.3. Detoxifying bee products to destroy chemicals
      7.6.4. Recommendations for sterilising and detoxifying bee products fed to caged adult workers in the laboratory
   7.7. Controlling for water evaporation from food provided to caged adult workers in the laboratory
   7.8. Feeding tests using caged adult workers in the laboratory

      7.8.1. Starving caged adult workers in the laboratory prior to performing a feeding test
      7.8.2. Feeding a liquid test substance to individual adult workers in the laboratory
      7.8.3. Feeding a liquid test substance to groups of caged adult workers in the laboratory
      7.8.4. Feeding a solid test substance to groups of adult workers caged in the laboratory
      7.8.5. Recommendations and considerations for oral exposure of a test substance to caged adult workers in the laboratory
8. Queens and drones
   8.1. Maintaining queens under in vitro laboratory conditions
   8.2. Maintaining drones under in vitro laboratory conditions
9. Conclusions and future directions
10. Acknowledgements
11. References