All of the early instar wax worms I have had in the 96 well plates were frozen and disposed of. After monitoring each well's survival for over two weeks I was not able to discern any sort of pattern, trend, or consistency in the type and concentration of spores they were exposed to.
The wax worms were exposed to B. thuringiensis and P. larvae spores on a single piece of oat. An image of the setup can be seen here. Based on my results using this setup it may not be the most effective way to expose the wax worms to spores. After 1 week the early instar wax worms had eaten about 1/2 of the oat and after 2 weeks those that were still alive had consumed most of the oat. The well may have no longer contained any oat, but it contained an almost equivalent amount of frass (insect feces). This could not be a very healthy environment for the wax worms.
Results & Discussion
The only big drop off in survival of the wax worms in the negative control (no spores) was seen on Day 1. Since this was only 24 hours after they were transferred to the wells their deaths can likely be attributed to handling mistakes (crushing with forceps). The deaths at Day 1 was significantly reduced in the 96 well plates that were started last, likely due to my improved skill with the forceps to transfer the wax worms without injuring them. Death in other treatment groups was also seen on Day 1, however those treatment groups seemed to experience more rates of death than the negative control after that day (which was expected).
I will not report on the exact day/deaths of each wax worm exposed to the different treatment groups as the results were highly inconclusive. There didn't seem to be much difference in the rates of death in wax worms exposed to the highest concentration of P. larvae spores (1000 spores) and those exposed to the lowest (1 spore). More surprisingly, there was not a difference in the wax worms that were exposed to B. thur (a known insect pathogen) spores compared to the other treatment groups. B. thur was used as a positive control, yet there was no difference in survival when compared to the negative control!
It is my belief that the reason for these sporadic results is due in large part to the method at which the wax worms are being exposed to the spores. After one week they only eat a portion of the oat and it is likely that the portion that is ingested doesn't even contain the spores that it was inoculated with. It is also likely that once the wax worm actually get around to ingesting the spores on the oat they may be at an instar that is able to fend off the disease. Although no research has been done to know at what instar the wax worms are/aren't susceptible to P. larvae, if at all. Additionally, since only a volume of 3 uL was added to the oat, the spores were only present on part of one side of the oat. It would be ideal to more evenly distribute the spores on the food source for the wax worm's ingestion, however that might not be possible with the oats. For these reasons, I would like to move on to a different food source in this study.
Using Wax and Honey
All of my wax worms in my colony are currently pupating, so I am unable to continue with the early instar LD50 study at this time.So, I worked on creating an alternative method (as opposed to the oats) for exposed wax worms to the spores. I would like to use either bee's wax or honey to replace the oat in this experiment. With either of these two types of diet I could much easier homogenize the spores and it would be a more natural route of exposure for the wax worms.
I would still like to use the 96 well plates, but I need to devise a way to coat the bottom of the wells with either wax or honey, preferably the wax. I am a bit concerned with the early instar wax worms drowning in the honey if there is too large a volume present. A small shaving of bee's wax (used a razor to cut) was transferred to a number of wells using a forceps. Additionally, a small volume of honey was also transferred to a number of wells. The 96 well plate was placed in a heated plate warmer in order to heat the plate and melt the wax/honey to the bottom of the well.
Results were varied, but over all not very promising. The plate needed to be heated to nearly 100 C to melt the wax into place. I am concerned I am getting near the temperature required to melt the polystyrene plate (something that has happened before using the same plate heater). I am also concerned with how the spores will handle such high temperatures. I will continue to fine-tune the wax process, perhaps by heating a batch of wax first and then transferring the hot wax via a pipette to the wells.
//EWW
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