Carbon dioxide

Exposure to carbon dioxide deprives individuals of oxygen, and depending on dose, can lead to anoxia or asphyxiation in various tissues, as well as the accumulation of acid metabolites that can impair physiological processes, especially in the nervous system (Nicolas and Sillans, 1989). Exposure to carbon dioxide can result in premature aging and reduced lifespan of worker honey bees (e.g., Mackensen, 1947; Austin, 1955; Woyciechowski and Moron, 2009), as well as affect behaviour and memory (Erber, 1975; Nicolas and Sillans, 1989). Although, exposure to carbon dioxide can influence intra-host parasite development (Czekońska, 2007), it is uncertain if honey bees exposed to the gas are subsequently more susceptible to parasitic diseases.

Phenotypic response to carbon dioxide is dose-dependent. Whereas large dosages and long exposure of carbon dioxide (i.e., >95 % for 105 minutes) result in significant mortality and behavioural changes (Rueppel et al., 2010), much shorter exposure duration can still affect workers. For example, pure carbon dioxide treatments greater than 15 seconds influenced sucrose response, foraging behaviour, and survival, although, in some cases certain symptoms may abate over time (Ebadi et al., 1980; Pankiw and Page, 2003). Similar to workers, queens receiving a carbon dioxide anaesthetic can also exhibit symptoms; for example, higher carbon dioxide: nitrogen ratios resulted in significantly earlier oviposition events (Chuda-Mickiewicz et al., 2012). More details on anaesthetizing queens can be found in the BEEBOOK paper on instrumental insemination (Cobey et al., 2013). To immobilise worker honey bees using carbon dioxide, researchers should provide individuals to pure gas for 10-15 seconds (Ebadi et al., 1980); this should render individuals unconscious for approximately 15-30 seconds.

Protocol to immobilise bees with carbon dioxide:

  1. Place honey bees in a well-ventilated cage.
  2. Transfer the cage to a sealable plastic container with a small opening in the lid.
  3. Place the caged honey bees at the bottom of the sealed container as an added precaution to ensure full carbon dioxide exposure (Ebadi et al., 1980), since carbon dioxide is heavier than air.
  4. Connect a tube to the gas source (carbon dioxide bottle).
  5. Insert the other end of the tube into the opening of the plastic container lid.
  6. Provide constant supply of carbon dioxide (e.g., 100 ml per minute) for 10-15 seconds.