Detection of P. larvae in Local Honey

From 3-17-15.

Repeated PCR of the honey isolates using the AFB primers. Ran 1% gel, 10 uL amplicon DNA + 2 uL EZ vision dye. Below is an image of the gel ran at 100 volts for 60 minutes.

Lane 1	Lane 2	Lane 3	Lane 4	Lane 5	Lane 6	Lane 7	Lane 8
Hi Lo	8a	2a	22a	63a	Q	R. Trach	9545

The template DNA used in this reaction is the same that was used in the previous PCR using the AFB primers only I used half as much as before. The positive control, P. larvae 9545, amplified using these P. larvae specific primers. The roach trachea (lane 7) served as a negative control as it has not amplified using these primers in the past and is highly suspected to a Bacillus spp. This time with the honey isolates you can clearly see the bands and there is minimal DNA left in the wells (no smearing). All of them amplifed except for 8a (Lane 2), which has amplified in the past indicating that it is indeed P. larvae . However, you can see that there is something in the well of this lane and it could be that I still used too much DNA for that isolate. In the future, I will only use half the amount of DNA that I have been using when setting up my PCR reactions.

//EWW

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

Detection of P. larvae in Local Honey

From 3-11-15.

Re-ran PCR from 3-11-15 using the AFB primers. The lanes are set up as before. 10 ul of ampicon DNA was combined with 2 uL of EZ vision dye and loaded into the well. The 1% gel was ran for 1.5 hours at ~100 volts. The gel is seen below:

Top:
Lane 1	Lane 2	Lane 3	Lane 4	Lane 5	Lane 6	Lane 7	Lane 8
Hi Lo	8a	2a	22a	63a	Q	R. Trach	None
Bottom:
Lane 1	Lane 2	Lane 3	Lane 4	Lane 5	Lane 6	Lane 7	Lane 8
	Hi Lo	TransConj	TransConj	TransConj	9545	Neg

Discussion:
The good news is that there were nice clean bands seen, the bad news is that there weren't a lot of them (which there definitely should have been). Even the positive control of P. larvae 9545 didn't have a band, which indicates something is clearly amiss. There appears to be a lot of DNA caught in the wells, which may be an indication I am using too much template DNA. Most of the template DNA I used was from a crude genomic prep (heating whole cells), so it is odd that they would result in such a high quantity of DNA. The entire top row was expected to amplify (except for the R. (roach) Trach (trachea) using these AFB primers as they have be shown to in the past. How odd that only a couple of them did. I seem to be having some trouble with PCR lately, it could be attributed to the fact I haven't done it in so long and have lost some of my finesse. I am planning on running more PCR in the near future, but if I am unable to even get my positive control to amplify I am going to have a hard time. So, I will double check my procedure, perhaps I am missing a crucial step or PCR ingredient, or perhaps my thermocycler program has been altered. I will try this applification again using less template DNA, likely diluted 1:10.

//EWW

Chlorine Dioxide - Wax Worm Lethality

From 3-16-15.

Checked on the wax worm health today (~18 hours after ClO2 exposure). Interestingly, the 10 wax worms that were exposed to the chlorine dioxide gas are now all dead and all the ones that were not exposed are still alive and well.

These results confirm my hypothesis, that the cholrine dioxide gas would kill the wax worms. The reasons I am using the gas was to gauge its efficacy at killing P. larvae spores, and possibly use it as a method to treat an American Foulbrood diseased honey bee hive. These results also don't bode well for the likelihood of the gas not killing the honey bee larvae.

All the wax worms were frozen at -20C overnight to kill them and then disposed of in a bio-hazard container.

I may do a dose titration experiment with varying concentrations of ClO2 gas and the wax worms in the future, depending on the quantity of wax worms I have available.

//EWW

Wax Worm/P. larvae LD50

From 3-14-15.

It has been seven days since the early instar wax worms were fed BAD spiked with P. larvae spores. Started on 3-3-15. The raw survival data is saved to an Excel file, but the data is also displayed in the two tables below. Statists were calculated as three replicates of 10 wax worms each.

Raw data results

Normalized data to give percent survival

Concentration of spores that were put onto each plate (taking into account the 100 uL volume)

1:5 = 2000 spores

1:25 = 400 spores

1:125 = 80 spores

The above concentration of spores were added to each container with 10 early instar wax worms in each container. So, it is likely that each wax worm only ingested about one tenth of the concentration above.

Discussion:

Early instar wax worms that were fed the only the BAD without spores had only about 78% survival after seven days, which isn't that bad, but not as good as it should be or I'd like it to be. I believe that the low survival is due to the small storage container that was used. There were 10 wax worms in each small container and the container eventually filled with feces and other debris. Interestingly, the wax worms will eat organic matter, including each other. This is especially evident when there are different sizes of wax worms present in the same container. On one day a wax worm was completely missing from the container when I checked survival. There was no sign of it anywhere and I am absolutely sure it did not escape, however, the container was full of early instar wax worms that were about three times the size of the missing wax worm. I cannot confirm its fate, but I believe it was eaten either to a diseased state making it more vulnerable, or just simply being a runt of a wax worm.

There was a trend of survival seen depending on the amount of spores that the wax worms were fed. The 1:5 dilution resulted in the most deaths and at the quickest rate and so on. However, the error bars indicate that this trait is not statistically significant, meaning there was no distinction between the amount of spores given (1:5 to 1:125) and the survival rate. It is also evident that I should have gone longer beyond seven days to check survival and will likely do so in the future.

I will attempt this experiment again in the future, hopefully with more early instar wax worms and a higher concentration of spores. I need to find a way to categorize the instars of the wax worms and then sort them for the experiment.


LD50 Injection
I checked the late instar wax worms that were injected with P. larvae and B. thur spores yesterday. All of the wax worms that were injected with B. thur are now dead. Pictures of what they looked like only ~18 hours after injection are seen below. They were non-responsive when prodded with a forceps and visibly look dark and deceased. These wax worms were injected with 4x10^5 B. thur spores, which is a very high concentration.

WW injected with Bacillus thuringiensis after one day.

WW injected with Bacillus thuringiensis after one day.

After the above wax worms were disposed of, I realized that maybe they were not dead, but in fact have begun of pupating... It could be that after injection of the B. thur spores triggered pupation in the wax worms. I will most likely repeat this experiment using B. thur as a positive control, albeit at a lower concentration.


//EWW

Chlorine Dioxide - Wax Worm Lethality

The Goal: Determine if chlorine dioxide gas is able to kill adult wax worms (5th instar) at any concentration.

Since I have plenty of wax worms and chlorine dioxide gas components I thought I'd give this a try. This may lead to an effective way to treat wax worm infected hives in the future.

Ten late instar wax worms were placed into two Tupperware containers (volume of 1.2 Liters). One of the containers was not exposed to the chlorine dioxide gas at all, this will serve as a negative control. Below is a picture of the 10 wax worms in the large container.

10 WW in Tupperware container that will be exposed to ClO2 gas

The other container of wax worms will be exposed to chlorine dioxide gas. Approximately 1.5 grams of Part A and Part B chlorine dioxide reagents was added to the small container seen in the picture below. The idea was to keep the wax worms away from coming into direct contact with the reagents and also allow the reagents to mix together. 

Created a container to mix the ClO2 components that the WW will not be able to com into direct contact with. It is wire mesh and a cover. The wire mesh is taped on the back.

The chlorine dioxide gas reagents were added and the above reagent container was added to the center of the 1.2 Liter Tupperware container. The container was covered with its lid and the edges were wrapped with Parafilm. The control and experimental containers were place at room temperature, protected from the light in the Fisher Lab. It is very important that the containers are protected from the light as it can effect the generation of chlorine dioxide gas.

I am fairly certain that the gas will kill the wax worms due to its lethal effect on smaller organisms and mode of killing. I also added a rather large concentration of reagents, so I will be surprised if it doesn't kill the wax worms. But, just to confirm this hypothesis I will test it out.

//EWW

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