P. larvae LC50 SOP

Below is the tentative protocol for determining the LC50 of P. larva during the first instar of honey bee larva.

Lethal Concentration (LC50) Assay of P. larvae in A. mellifera (Honey Bee) Larva

Protocol to infect 1^st instar larva with P. larvae

At USDA

1. Trap queen honey bee inside Jenter box. Place box inside bee hive and leave queen trapped for 24 hours. (Monday)
2. Remove cap cover of Jenter box so that the queen can escape. Place box back into bee hive. Leave box inside hive for 72 more hours. (Tuesday)
3. Remove Jenter box from hive and transfer to USDA building covered by a moist cloth inside a Styrofoam container. (Friday)
4. Carefully transfer honey bee larva from the Jenter box to 10 uL of Bee Artificial Diet (BAD) in 24 cell culture plates using a camel hair paint brush. Transfer one larva to one 10 uL puddle of BAD in one of each of the wells.

Note: Try to select only the smallest larva for 1^st instar development stage.

5. Transfer bee larva in a Styrofoam container to the Fisher lab in Van Es at NDSU.

At NDSU

6. Dilute small portion of BAD with different concentrations of P. larvae spores from Spore Stocks stored in 4C to create the following five treatment groups:

Treatments 200 CFU/mL 400 CFU/mL 1000 CFU/mL 10,000 CFU/mL No CFU/mL (control)

7. Pipette 5 uL of each treatment group into 24 well plates and carefully transfer bee larva from Step 5 to each of the newly created BAD-P. larvae puddles (one larva per 5 uL). Incubate plate at 35C for 24 hours.

Note: Ideally, n = 100 for each treatment group.

8. Check survival of each bee larva using the dissecting microscope. Live larva will slightly ungulate and appear to be a light white/cream color. If dead- remove from experiment, record weight, and free at -20 for future use. If alive – move to Step 9.
9. Transfer experimental treatment group larva to a new 24 well plate containing 10 uL of un-inoculated BAD. Add 10 uL to control larva. Incubate at 35C for 24 hours.
10. Continue to check survival for a total of nine days. Continue to provide larva with increasing amounts of BAD as described in the table below:

Day	BAD provided
1	5 uL
2	10 uL
3	20 uL
4	30 uL
5	40 uL
6	50 uL
After 6 days, cease BAD

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