Wax Worm/P. larvae LD50

From 3-31-15.

It has been seven days since the wax worms were exposed to P. larvae spores both through injection and through their diets. I have been closely monitoring their health and survival. When a wax worms had died in any of the groups it was removed.

LD50 Injection

There were three biological replicates for each treatment, with ten technical replicates each. The below graph was made using Prism 6.0, however it does not include error bars. I wanted to test my skills at using the Prism software and found it to be surprisingly easy to use- you can just copy and paste your data from an Excel spreadsheet (where I had it) to Prism.

Average number of WW that were alive at each time point (n=10)

There was no deaths recorded in both the "No Inject" control group over the course of seven days. There was minimal death observed in the PBS injected group, which I attribute to poor injection skills- this is only about the second time I've injected WW. It is possible that some sort of internal damage was caused by me sticking the needle too far inside the WW to inject. Unfortunately, the groups of wax worms injected with 2000 P. larvae spores showed similar survival to those injected with PBS. This is the same trend that has been seen previously (at least it's consistent!). I likely need a higher concentration of P. larvae spores to inject the WW with in order to cause disease and death... if P. larvae causes disease in WW. If the WW is injected with a high enough concentration then simply the sheer volume of bacteria would be enough to cause the death of the WW. As previously seen from the images I've posted, there are potential signs of disease in the wax worms that have been injected with P. larvae, however it does not lead to their death like what is seen in the B. thur spore groups. The positive control in this experiment made use of the known insect pathogen B. thuringiensis spores. WW were injected with 200,000 B. thur spores and just after the first day about half of the injected WW were dead. This trend is also what has been previously seen. The problem remains, as always, I need to have access to higher concentrations of P. larvae spores.


LD50 Ingestion

Spores added to the container with 5 wax worms:

P. larvae = 10,000 CFU

B. thuringiensis = 1,000,000 CFU

For this study, early instar WW were fed Bee Artificial Diet (BAD) spiked with bacterial spores. A similar overall trend was seen with the WW in this study that was seen in the LD50 Injections study as well. Minimal deaths were observed in the No Diet and No Spores in Diet control groups. The WW that were fed the P. larvae spores had a similar survival that was seen in the two control groups. Once again, a large amount of deaths was recorded in the B. thur group. By day three all the wax worms in this group were dead in all three of the replicates. This isn't overly surprising as the WW were exposed to a very high concentration of B. thur spores. I stopped the experiment after seven days since survival seems to have leveled off in all of the groups, indicating that likely if the spores haven't led to the death of the WW- they never will.

//EWW