Wax Worm/P. larvae LD50

From 3-9-15.

Day 5
Checked on the early instar wax worms that were fed the P. larvae spores. On day 4 they began to show visible signs of illness on their bodies. This is about the time frame as before. Images of these black/brown sores on their bodies can be seen below. I have also begun to see an increased rate of death in the less dilute spore groups.

Early instar WW with signs of illness (sores on body)

Dead WW that was exposed to the 1:5 P. larvae spore dilution in its diet 5 days ago.

Dead early instar WW. Notice deformity in body were the sores are.

LD50 Injections

I plan on determining the LD50 of P. larvae spores in the later instar WW as well. For the later instars I plan on injecting the spores directly into their hemocoel.


I injected late instar WW with 20 uL of a diluted spore stock using a small needle. I was able to hold the larger WW in place and inject them near the second to last segment of their body (second from anus end).

For a positive control I used B. thuringiensis, a known insect pathogen, and for a negative control I used PBS. For the experimental groups of P. larvae I used three different dilutions (5 fold serial dilution). Below is the amount of spores that was actually injected into each of the wax worm (accounting for dilution and the 20 uL volume that was injected):

Bacillus thuringiensis - 4x10^5 CFU (spores)

P. larvae - 4x10^2 CFU, 8x10^1 CFU, and 16 CFU

Phosphate Buffered Saline (PBS) 1x

WW after injections, stored in petri plate (10 per plate).

The area were the WW were to be injected were wiped with ethanol and allowed to briefly dry as to avoid any secondary infection (I work in an insect pathology lab!). The injections themselves took place on the lab bench on top of a standard white sheet of printing paper. The paper was switched between treatment groups as to avoid cross contamination that would effect the outcome. 

Comments on injecting WW: Be confident with the needle stab and be sure to insert the needle far enough in that the 20 uL volume doesn't leak out due to a superficial stab. After injecting the volume, let go of the wax worm with the needle still inside of it and then pull out the needle. This prevents putting physical pressure on the WW that would cause the injected volume or hemolymph from coming out of the newly created injection wound.

After injection, the WW were carefully transferred to a petri plate either via careful use of forceps or by rolling. Each treatment group included 30 WW (10 to a petri plate). The WW were placed inside a secondary container and stored in the 30C walk in incubator.

LD50 Diet

Late instar wax worms were also allowed access to Bee Artificial Diet (BAD) that had been spiked with P. larvae spores. No B. thur control was used in this experiment as I am running low on BAD and am not convinced this particular experiment will go anywhere, but we'll see. I am unsure if the late instar WW will even ingest the diet if not force fed and I am also not sure that if they do indeed ingest the spiked diet if the spores will have an affect on their now more functioning immune system. If it does, I also believe it would likely require a higher concentration of spores in their diet to overwhelm their immune system and cause disease. So, the results of this experiment may not tell us anything if the WW all survive, but if they don't then that would encourage definite further investigation. Plus, I had extra BAD spiked with spores from the last experiment and plenty of wax worms to try something with.

Ten WW were sorted into a single petri plate and fed the P. larvae spores in BAD at a volume of 200 uL. The 200 uL were added to the plate in the fashion shown below in the image. This was to prevent drowning of the wax worms in one large puddle of diet as well as to increase the chance that a WW will come into contact with the diet (more spots = better chance)

WW fed 200 uL of P. larvae spore-spiked BAD

P. larvae spores on the plates (accounting for the 200 uL volume actually added):

1:5 dilution of stock = 4000 CFU (spores)

1:25 dilution of stock = 800 CFU

1:125 dilution of stock = 160 CFU

The WW were placed into a secondary container and stored in the 30C walk in fridge.

I will continue to monitor the survival and health of the all the LD50 wax worms for at least a week depending on how survival goes.

//EWW