Wax Worm/P. larvae LD50

From 3-24-15.

Set up the P. larvae spores LD50 ingestion and inoculation experiment using the early and late instar wax worms larvae again. Unfortunately, I used up the last of my P. larvae spore stock reserves and will need to replenish, hopefully the use of Columbian sheep's blood agar to improve sporulation is successful. I am also running low on the Bee Artificial Diet (BAD) and will have to acquire more from the USDA soon.

LD50 Ingestion

The smallest wax worms (earliest instars) were added to the small containers used previously for this experiment. However, this time only 5 early instar wax worms were added to each container instead of 10. This is in response to the universal decline in health seen in all the wax worms last time due to possible overcrowding and excess frass build up. A volume of 100 uL of BAD spiked with P. larvae spores was still added as before. With the same volume of spores, but reduced amounts of present wax worms, will likely increase the amounts of potential spores ingested by each worm.

Spores added to the container with 5 wax worms:

P. larvae = 10,000 

B. thuringiensis = 1,000,000

100 uL of BAD was distributed throughout the container to avoid large puddle that WW could potentially drown in. By spreading it throughout also increases the likelihood the WW will come into contact with the spores.

B. thur was used as a positive control and was also mixed with BAD when made available to the wax worms. Two negative controls were also implemented, one group was fed only the BAD and another was not provided a diet at all. The containers were sealed with their covers and incubated at 30C in a secondary container. I will check their progress and survival everyday.

LD50 Inoculation 

The larger wax worms were inoculated with 20 uL of P. larvae spores in PBS. Injection spots were near the second to last segment of their body into their hemocoel as done previously. B. thur was used as a positive control. Two negative controls were also used, one set of wax worms were injected with only 20 uL of PBS and another was not injected at all. This will be important to see if making cocoon trends occur between all the groups. Ten wax worms were added to a single petri plate and incubated at 30C in a secondary container. I will continue to monitor their survival in the coming days.

//EWW