4.1.1. Protocol for exposure bioassays

  1. For grafting of larvae, dispense 300 µl of larval feed per well in a 24-well plate. Leave 6 wells empty (A1, A3, A5, D2, D4 and D6) and fill them with 500 µl of double distilled water to avoid desiccation.
  2. Incubate plates 30 min at 35°C to warm them up.
  3. Graft the larvae by inserting a grafting tool under the back of the larvae floating in royal jelly without touching it, and carefully deposit it on the surface of the larval feed prepared in the well plate. Deposit ten larvae per well.
    Note: Graft only L1 instar larvae, under 12 hours of age from hatching. Collecting larvae from as many different populations as possible will ensure randomization, achieve homogenous treatment groups, and avoid population-specific variations.
  4. For starting the experimental phase, aliquot larval feed and add the necessary volume of spore suspension, which needs to contain a defined concentration of spores to adjust a defined final spore concentration in the larval feed, e.g., to the desired lethal concentration (LC) for the infection group (Table 4).
  5. Dispense 300 µl per well of the spore-contaminated larval diet in 3 different wells. Leave 3 wells for the control group receiving non-spore contaminated larval diet during the entire experiment.
  6. Set ten larvae per well.
  7. Incubate plates at 35°C for 24 hours.
  8. Groups of 30 larvae (in 3 wells) are treated as one replicate and at least three independent replicates should be performed for statistical analysis (see the section on statistics in the BEEBOOK paper on miscellaneous methods (Human et al., 2013)).
  9. After 24 hours of infection, transfer larvae to a pre-warmed, fresh normal larval diet plate.
    Use a different grafting tool for each treatment group to avoid reinfection. Thereafter, every treatment group receives fresh larval diet every 24 hours.
  10. Analyze the plates each day under a stereo microscope to determine the health status of the larvae.
  11. Transfer remaining (living) larvae to a new plate containing pre-warmed fresh larval diet.
  12. Proceed with experiment until day 14.
    Since larvae increase in size during the experiment, the number of larvae per well must be decreased accordingly.
  13. After defecation (at day 7-8, when light yellow secretion can be observed surrounding the larvae), transfer larvae to pupation plates. Prepare pupation plates by lining every well with laboratory tissues, leaving 6 wells free for double distilled water.
  14. Larvae are classified as dead when they stop breathing (movement of tracheal openings stops) and lose body elasticity. The number of dead larvae should be reported every day.
  15. To determine whether P. larvae infection caused the death of a larva, dead larvae are plated out on Columbia sheep blood agar (CSA) plates. Plates are incubated overnight at 37°C to allow the growth of vegetative bacteria only (spores need about 3 days to germinate under these conditions). Positive AFB infection will be confirmed by growth of P. larvae (see section 3.2.).
  16. Further confirmation is provided by performing P. larvae-specific PCR-analysis of colonies grown from larval remains.
    16.1. Pick one colony.
    16.2. Dissolve it in 25 µl of double distilled water.
    16.3. Boil at 95° for 10 minutes.
    16.4. Centrifuge for 10 minutes at 9,500 rcf.
    16.5. Supernatant can be used as template for PCR (see section 3.2.2.).

Table 4. Estimated LC50 and LT100 values (min-max) for ERIC I and ERIC II. (Genersch et al., 2005).


LC50 (CFU ml-1 larval diet)

LT100 (days p.i.)