220.127.116.11. Individual feeding
Many Nosema laboratory experiments individually feed bees with an inoculum to ensure that bees are exposed to a known quantity of spores (e.g. Higes et al., 2007; Paxton et al., 2007; Maistrello et al., 2008; Forsgren and Fries, 2010). The volume of the inoculum can be 5 to 10 ml, amounts that are readily consumed by bees. Individual feeding of spores produces significantly lower variation in response infection level compared to group feeding of spores (Furgala and Maunder, 1961) and should be preferred for most experimental purposes until further investigations comparing group versus individually fed honey bees occurs. As with other laboratory procedures concerning Nosema, it is important to minimize chances of contamination by setting up sterile feeding stations unique to each treatment group. To individually inoculate honey bees with Nosema spores, refer to the BEEBOOK chapter on maintaining bees in vitro (Williams et al., 2013).
Specifically for Nosema studies, experimental bees should be starved in hoarding cages for two to four hours by removing all feeding devices to ensure that the entire inoculum is ingested quickly (e.g. Fries et al., 1992; Malone and Stefanovic, 1999; Higes et al., 2007; Maistrello et al., 2008). Bees destined for each treatment group should be maintained in separate hoarding cages to avoid the possibility of contamination during the removal of individuals for feeding. Because feeding can require considerable time depending on the number of bees to be inoculated and the number of persons available to feed spores, it may be necessary to house bees for each treatment group in multiple cages that can be starved in different time blocks to ensure that all individuals are without food for a similar period of time. Pre-trials will determine how much time is required for feeding, but generally it should take one to two minutes per bee. Additionally, and depending on the time required to feed all experimental bees, it may be prudent to rotate feeding amongst treatment groups so that, for example, not all bees from one treatment group are fed in the morning whereas bees from a second treatment group are fed in the afternoon. See previously discussed subsections within section 2.2.4., as well as section 2.2.7., for creating and choosing specific spore suspensions.
Once inoculated, bees can be placed into a 1.5 ml microcentrifuge tube with a breathing hole at the end in suitable growth chamber conditions in detail below for 20 to 30 minutes to ensure that spores are not transferred among bees (see Williams et al., 2013) (Kellner and Jacobs, 1978; Verbeke et al., 1984). Afterwards, the bee can be placed in an appropriate treatment hoarding cage. The provided description can be applied to inoculating all castes. However, drones and queens can be transferred to appropriate treatment cages immediately after feeding because they are only recipients during trophallaxis (Crailsheim, 1998)..Future studies should determine whether this is required as some debate exists regarding the frequency that newly emerged bees in cages engage in trophallaxis (Crailsheim, 1998).