Wax Worm/P. larvae LD50

From 4-4-15.

Goal: Design a new method to use in determining the LD50 of the early instar wax worms to P. larvae spores.

Background: One of the issues I was having with the previous experiments was that the wax worms were not fed a high enough concentration of spores and I never actually was able to kill them. The B. thur spores were able to cause death in the wax worms after about three days, but all the P. larvae exposed wax worms still survived (or died due to other causes). However, signs of disease was observed in some of the wax worms exposed to P. larvae by day three, but cleared up by day six. The main reason that the early instar wax worms were not fed a high enough concentration of P. larvae spores was because my titers were not very high. Fortunately, I have increased my titer of P. larvae spores through use of the Columbian sheep's blood agar. The titers have increased from ~10^3 to ~10^6.

Another issue I had ran into previously was with storing numerous wax worms together that were in the same treatment group. The main concern with this was that frequently I would count surviving wax worms after each time point and there would be one completely missing from the container. I am extremely confident that the wax worm did not escape. My conviction in this statement was cemented after several small pieces of a wax worm's head was found inside the container (missing it's body). This was observed several times and upon further investigation I discovered that the wax worms will actually eat each other! They will eat organic material, including each other if the food source is scarce, which was also true since they were only provided the BAD spiked with spores as nutrients.

Also, there was some (more than I'd like) death in the early instar wax worms at Day 1. This was often attributed to either handling issues, which became reduced once I become more experienced moving the wax worms, or drowning of the wax worms in the diet. There were a number of wax worms that would be floating in the diet at Day 1. There was only a small volume pipetted into the container (between 5-10 uL), however they are VERY small early instar wax worms.

The last big issue I had with the previous experiment was the type of containers that was being used. There was no air transfer using those containers, and even though they were opened daily to count survival, humidity was noticeably building up inside the container. I am not sure how much oxygen an early instar wax worms needs for 24 hours, but I am pretty sure with 5-10 of them inside the small tightly sealed container, there wasn't enough. Coupled with the large amount of wax worms per container (between 5 -10 depending on the experiment) and the lack of air exchange the containers themselves became very inhospitable by the end of the experiments. 

Method: For this experiment, we wished to expose the wax worms to a higher concentration of P. larvae spores (in a decreased volume), store them individually in a 96 well plate, and use a breathable cover for the plate to facilitate air exchange. The next generation of wax worms have just began to hatch from their eggs a few days ago and are now a size that is easy to work with for this experiment.

1. Add a single piece of oat meal to the bottom of a well in a 96 well plate (I am not 100% sure if they WW will eat the oatmeal, but it is meant to serve as their nutrient source over the course of this experiment.
2. Serially dilute P. larvae spore stocks in sterile ddH2O and add a volume of 3 uL directly to the top of the piece of oatmeal inside the well. Allow the liquid to be absorbed into the oatmeal (~1 minute).
3. Add an early instar wax worm is then carefully transferred to the well using a forceps. Make sure it doesn't escape the well before it is covered.
4. Cover the wells using a metal sticky cover seal. Create a very small pin sized hole in the top of the film for the wax worms to breath and allow air to be exchanged.

The smiley bug is obviously a wax worm

5. Once all the wells are covered, tip the 96 well plate upside down and place inside a secondary container. Incubate the container at 37C and check survival of the wax worms every 24 hours.

Note: the reason the plates were not covered with a more permeable (or breathable) cover is because the WW will eat that material and escape the well. This is also the reason the plates were tipped upside down- to prevent them from leaving their wells.

For this experiment,

Control:
Negative - sterile ddH2O
Positive - B. thur spores (stock conc of 10^8 CFU/mL)

Experimental treatments:
P. larvae spores- stock (2.1x10^6 CFU/mL) and dilutions: 10^-1, 10^-2, 10^-3, 10^-4
Meaning the wax worms were fed 1000 spores, 100 spores, 10 spores, 1 spore, and essentially no spores (0.1 spore).

//EWW