Rearing and Injection of Manduca sexta Larvae to Assess Bacterial Virulence

The overall goal of this procedure is to assess bacterial pathogenicity against insect larvae using manduka seca as a model organism. This is accomplished by first rearing the insect larvae from sterilized eggs to the fourth instar phase of development. In the second step, the bacterial samples are prepared for injection.

Next, the bacteria are injected into the fourth instar manduka seca larvae. In the final step, the death of the insects Over time after injection is monitored. Ultimately, the time to insect death is recorded and used to assess bacterial virulence.

This method can help address key aspects in the symbiosis field, including the discovery of novel insect pathogens and determining differences in virulence among bacterial species or strains. Begin by first autoclaving, 15 grams of auger in 900 to 1000 milliliters of water. Immediately after autoclaving.

Mix the auger with 166 grams of wheat germ diet. In a laboratory blender, pour the blended insect diet into a dish. When cooled, transfer the diet from the dish to a piece of aluminum foil.

Wrap it tightly and store it at four degrees Celsius. Upon arrival, place the M sex to eggs in a glass filter holder and vacuum flask apparatus with a 90 millimeter filter paper. Then pour 250 milliliters of 0.6%bleach solution onto the eggs for two to three minutes stirring occasionally.

Now turn on the vacuum to drain the bleach solution, and then wash the sterilized eggs three to four times with 250 milliliters of sterile distilled water per wash. Next, place a fresh 90 millimeter piece of filter paper into a Petri plate lid, and then transfer the eggs to the filter paper to allow them to dry in a flow hood until they no longer stick together. Now place the recently prepared insect diet on top of a secured rubber stopper in several plastic containers and transfer approximately 40 eggs into each container, separating the eggs from the diet to prevent moisture induced fungal contamination.

Maintain the eggs at 26 degrees Celsius with a 16 hour light, eight hour dark photo period. The eggs will lighten to a yellow white color prior to hatching when the hatching is complete. Using forceps to pick up each of the larvae by the long black horn, carefully transfer approximately 25 larvae each to new containers with diet at the bottom.

Incubate the larvae for two days under the same conditions as for the eggs, while larval death is unusual at this stage, some dead or more likely developmentally stunted larvae may be observed. Now, transfer the larvae to individual containers with small pieces of food to avoid cannibalistic behaviors and incubate them again. Replace the food and clean the feces out of the containers every other day until the insects undergo the third larval molt.

The third larval molt is the fourth instar larval stage characterized by the appearance of black hook like crochets on each prole and by an increased prominence of the stripes along the insect body. After growing out the desired bacterial strains to be tested, spin down 500 microliters each of the bacteria strains for two minutes in a micro centrifuge at 17, 000 times, G at room temperature. Then wash the pellets in one milliliter of sterile PBS under the same conditions.

After resus suspending the cells in another half milliliter of sterile PBS measure the optical density of the suspensions before beginning the injections using a fresh pipette tip for each dilution, prepare six serial tenfold dilutions of the first bacterial strain in sterile PBS in a 96 well microtiter plate. Now spot 10 microliters each of dilution, 10 to the minus two through 10 to the minus six along the top of a Petri plate containing the appropriate growth medium. Then tilt the plate so that the spots spread to the center.

Next, after removing the insect diet and feces, place the insect containers on ice for approximately five minutes while the insects are cooling down. Rinse a syringe needle three times each in two tubes of 70 to 100%ethanol, followed by one tube of sterile water. Then use a hemo cytometer to quantitate the diluted cell suspensions under a microscope.

Depending on the desired inoculum, draw 10 microliters from the appropriate microtiter. Well then swab the surface of the insect to be injected With 95%ethanol. It is most difficult to hold the insect in the appropriate position for injection, while also manipulating the needle and taking care not to puncture the insect gut.

Also, be careful to work quickly so that the insect doesn't warm up and begin to squirm while you're holding it for injection. Inject the 10 microliters cell suspension into the insect at less than a 45 degree angle behind one of the abdominal pro legs. Taking care not to puncture the gut, inject the contents just underneath the epidermal layer.

After injecting each insect with the first strain of bacteria, once again, plate the 10 to minus two through 10 to minus six dilutions this time into the middle of the plate. Let them flow toward the bottom of the plate. Now incubate the plates at the appropriate temperature, and then count the colonies to enumerate the inoculum.

After repeating the dilution, plating, and injection process for each strain of bacteria, inject three to five insects with sterile PBS for the negative control group. Then incubate all the insects at 26 degrees Celsius and the 16 hour light eight hour dark photo period. Finally, monitor the insect survival over time after the injections, the insects often exhibit appetite loss, watery, yellow diarrhea, excrement and or water loss during infection prior to death.

Insect death is characterized as a lack of voluntary movement upon stimulation. In this assay, the insects were injected with approximately 50 colony forming units of either wild type or an attenuated mutant strain of Xer Haus NLA groan to mid log phase. The insects were observed for approximately 72 hours and the percent of the injected insects that were still alive at each time point was recorded.

In this experiment, the attenuated strain exhibited a clear delay in insect killing, whereas the wild type strain killed all six larvae within 30 hours post-injection prior to the death of any of the mutant infected larvae. The data are statistically distinct Following this procedure. Other methods such as RNA extraction and quantitative real-time polymerase chain reaction can be performed in order to answer additional questions such as determining whether or not the bacterium is able to manipulate the insect immune response.