Wax Worm/P. larvae LD50

Goal: Create and LD50 curve of P. larvae spores using early instar wax worm larvae. This information will be important in determining if wax worms can be used as surrogates to the honey bee larvae. This information will also be used in a future grant proposal. 

The P. larvae spore stock G (1-31-15) that has since been stored at 4C in ddH2O was serially diluted 1:5 in Bee Artificial Diet (BAD) made last summer. The spores were diluted down to 1:3125. 

To set up the inoculation, the wax worms will be attempted to be fed the spores through the BAD. For this, small plastic containers (seen above) with covers will be used. About 150 mg of wax pellets were added to the center of each container using a metal scoopula. A volume of 100 uL of the diluted spores in BAD was added to the top of the wax pellets and mixed together using a sterile toothpick (picture seen below).

Mixing in the BAD/spores with a tooth pick

A total of 10 wax worms were added to each container. There were duplicate containers made of each spore dilution (2 biological replicates with 10 technical replicates each). Negative controls included a "no diet", where the wax worms were placed only on the wax pellets, and a "no bacteria", where the wax worms were introduced only to the BAD with no spores. Bacillus thuringiensis spores (a known insect pathogen) was used as a positive control (diluted 1:5 and 1:25).

10 WW to a single container

The containers were placed inside a tupperware container and placed in the walk in 30C incubator (where the wax worms were originally taken from). I will check the survival of each of the containers over the course of a week and hopefully be able to put together an spore ingestion LD50 if this is indeed a viable method of determining.


//EWW