1.5.1. Infection bioassays using in vitro rearing of larvae
Difficulties associated with controlling experimental conditions using honey bee colonies prompted the development of in vivo rearing conditions that allow rearing honey bee larvae in the laboratory and also a way to test the effects of pathogens, toxins and drugs. Refer to the BEEBOOK paper on larval rearing for a detailed protocol (Crailsheim et al., 2013). Exposure bioassays of in vitro reared larvae have been used to test differences in virulence of various A. apis strains (Vojvodic et al., 2011b), to compare the temperature response of chalkbrood and stonebrood (Vojvodic et al., 2011a), to test susceptibility of various honey bee subspecies towards A. apis (Jensen et al., 2009b), to test virulence of A. apis with or without presence of other Ascosphaera species commonly associated with solitary bees (Vojvodic et al., 2012) and to explore the host response to infection of A. apis at the molecular level (Aronstein et al., 2010).
Ascosphaera apis spores have to be ingested by the larvae, hence they must be incorporated in the larval food. There are two ways spores can be administered - either the larvae can be fed a small quantity (5 µl) of spore contaminated diet, which they will ingest quickly, or the larvae can be fed spore-contaminated diet ad libitum for a certain period. The advantage of feeding small quantities is that the exact dose per larvae can be controlled, and if fed with different dosages an exact LD50 can be calculated (Jensen et al., 2009b). Depending on the larval ages, additional feeding has to be done on the same day, approximately 2 hours later, after the first 5 µl is consumed. However if the aim is to produce a high numbers of infected individuals it can be more efficient to use surplus diet, as it is less time consuming.
All larval instars can be infected by A. apis (Jensen, unpublished), but it is recommended to use 2nd-4th instar larvae. When using 5th instar larvae, the A. apis spores have a limited time period in the gut for germination and penetration of the gut wall before the bee defecates, and 1st instar larvae are very fragile.