Wax Worm/P. larvae LD50

From 3-16-15.

LD50 Injection

Day 7 after injection and feeding of P. larvae spores to the late instar wax worms.

Below is the results of the WW injection LD50 study:

		3/14/15	3/15/15	3/16/15	3/17/15	3/18/15	3/19/15	3/20/15	3/21/15
	Injected:	Day 0	Day 1	Day 2	Day 3	Day 4	Day 5	Day 6	Day 7
P. larvae	400	10	10	10	10	10	10	10	9
	400	10	10	10	10	10	10	10	10
	400	10	10	10	10	10	10	10	10
	80	10	10	10	10	10	10	10	10
	80	10	10	10	10	10	10	10	10
	80	10	10	10	10	10	10	10	10
	16	10	10	10	10	10	10	10	10
	16	10	10	10	10	10	10	10	10
	16	10	10	10	10	10	10	10	10
B. thur	400000	10	0	0	0	0	0	0	0
	400000	10	0	0	0	0	0	0	0
	400000	10	0	0	0	0	0	0	0
PBS	None	10	10	10	10	10	10	10	10
	None	10	10	10	10	10	10	10	10
	None	10	10	10	10	10	10	10	10

At Day 1: all the positive control Bacillus thur. WW were dead as seen in 3-16-15 post.
At Day 2: I changed the petri plates that all the wax worms were in since there were getting very filthy with frass, the WW were also showing signs of disease (black spots) in the higher spore concentrations
At Day 3: Some have begun to form cocoons
At Day 5: black spots are no longer present in WW afflicted with them earlier
At Day 6: all of the WW are now in cocoons

Discussion:

The positive control WW died right away at Day 1, which is good because it shows that my ability to inject the WW was successful. However, this was an incredibly high concentration of B. thur spores relative to my P. larva spores. It could be related to the shear number of bacterial spores that I injected that caused death and not due to the pathogenesis of the bacteria. But, B. thur is a known insect pathogen. Regardless, the positive control fulfilled its purpose and I will continue to use B. thur as that control for the wax worms.

At day 2 the WW injected with a higher concentration began to show sign of potential disease with the formation of black spots on their bodies similar to what was seen in the early instar wax worms that were fed diet spiked with spores. However, this was about the time the petri plates were covered in copious amounts of frass from all ten of the wax worms and the signs of illness could have been a symptom of their inhospitable environment.

The WW all began to form cocoons as the time progressed. This phenomenon was not seen in the below experiment using the same instar of wax worms except they were fed a spore-spiked diet and not injected. After six days post injection all the wax worms in all the groups (including the PBS negative control) had formed cocoons in the petri plates.

Unfortunately, there was almost no deaths observed in any of the experimental groups (except one in the 1:5 which didn't appear to succumb to a disease). This does not bode well for identifying an LD50 curve. I obviously need to increase the concentration of spores that the WW are injected with, which has been an issue in the past as well. I am working towards an alternative method to culture P. larvae using columbian sheep's blood agar and BHI broth to increase spore yields.

I will re-attempt this experiment in the very near future, but am waiting on WW to grow in size enough to inject. The WW used in this experiment are still in the 30C walk in incubator and I will continue to monitor their survival until they pupate into wax moths in which case they will be disposed of.

LD50 Ingestion

It has been seven days since the late instar WW were fed BAD spiked with P. larvae spores in a volume of 200 uL for 10 WW in a petri plate. The survival was monitored closely for that time. Below is the results of this experiment:

		3/14/15	3/15/15	3/16/15	3/17/15	3/18/15	3/19/15	3/20/15	3/21/15
	Fed	Day 0	Day 1	Day 2	Day 3	Day 4	Day 5	Day 6	Day 7
P. larvae	4000	10	10	10	10	10	10	9	8
	4000	10	10	10	10	10	9	9	9
	4000	10	10	10	10	10	9	8	8
	800	10	10	10	10	10	9	8	8
	800	10	10	10	10	10	10	8	8
	800	10	10	10	10	10	9	9	8
	160	10	10	10	10	10	9	7	6
	160	10	10	10	10	10	8	6	6
	160	10	10	10	10	10	9	8	7
BAD	None	10	10	10	10	10	7	6	5
Only	None	10	10	10	10	10	8	7	5
	None	10	10	10	10	10	8	8	6

Discussion:

I did not change out the petri plates as I did with the LD50 Injection study above. There was a general trend of death starting at Day 5 that is seen in all the treatment groups, including the negative control BAD only group. There was no difference between the negative control and experimental groups in this experiment. I likely will not repeat the LD50 ingestion using the late instar WW in the future.

//EWW