2.2.4.1. Spore source

As with other parasites (Ferguson and Read, 2002), including N. bombi in bumble bees (Bombus spp.) (Tay et al., 2005) and bacterial diseases in honey bees (Genersh et al., 2005; Charriere et al, 2011), it is possible that genetic variants detected in both N. apis and N. ceranae (Williams et al., 2008; Chaimanee et al., 2010; Sagastume et al., 2011) may at least partially explain differences in host susceptibility and parasite virulence. Future studies should seek to identify genes responsible for Nosema epidemics (Chen and Huang, 2010). Based on differences in reported pathology of Nosema species around the world, researchers should state the region and country spores originated from. It is recommended that spores be sourced from multiple colonies.     

When creating inoculums, it is important to use fresh spores because their viability is lost over time when spore suspensions are stored. In particular, this is true for N. ceranae isolates, which rapidly lose viability in the refrigerator and almost completely lose infection capacity after freezing of spores (Fenoy et al., 2009; Forsgren and Fries, 2010). See section 2.2.5. for viability test procedures. Group feeding of caged bees using crushed bees infected with the respective Nosema spp. in sugar solution is a good way to propagate spores for experiments (see sections 2.2.4.4. and 2.2.4.5. for details on individual and group feeding, respectively). After 10-12 days, spore-inoculated bees from cages maintained in appropriate conditions (section 2.2.3.4) can be extracted and the ventriculi used for preparations of spore suspensions according to methods described previously (section 2.2.1 and 2.2.4.2). Molecular assays previously described (section 2.2.2.3) should be employed to ensure the proper species of spore is used for inoculations because mixed infections occur, even in naturally-infected individual bees (Burgher-MacLellan et al., 2010). Prepared spore suspensions can be used the next day when kept in sugar solution in the refrigerator (section 2.2.4.2.3)