Rearing and Injection of Manduca sexta Larvae to Assess Bacterial Virulence

1. Insect Egg Sterilization and Rearing

1. Prepare diet by first autoclaving 15 g of the provided agar in 900-1,000 ml H₂O. Immediately after autoclaving, mix with 166 g wheat germ diet and blend well in a laboratory blender. Pour into a dish (or dishes) to cool, then transfer diet to aluminum foil, wrap tightly, and store at 4 °C.
2. Upon arrival, sterilize M. sexta eggs with 250 ml of 0.6% bleach solution for 2-3 min in a glass filter holder and vacuum flask apparatus with a 90 mm filter paper, stirring occasionally.
3. Turn on vacuum to drain bleach solution and wash eggs 3-4 times with 250 ml sterile distilled H₂O per wash.
4. Transfer eggs to fresh 90 mm filter paper placed in a Petri plate lid and allow them to dry until they no longer stick together (about 20-30 min).
5. Transfer eggs to the bottom of plastic containers with insect diet, placing approximately 40 eggs in each container (Figure 1A). Eggs are separated from diet to prevent moisture-induced fungal contamination. Maintain eggs at 26 °C with a 16 hr light: 8 hr dark photoperiod. Eggs will lighten to a yellow-white color prior to hatching.
6. When hatching is complete, carefully transfer approximately 25 larvae each to new containers with diet on the bottom (Figure 1B). It is important to pick up each larva by the long black horn using forceps to avoid harming them during their fragile early developmental stages. Incubate for 2 days as above. While larval death is unusual at this stage, some dead or, more likely, developmentally stunted larvae may be observed. Such insects should be excluded from further study and sacrificed by freezing.
7. To avoid cannibalistic behaviors, transfer larvae to individual containers with small pieces of food (Figure 1C) and incubate as above. Monitor insect development, replacing food and cleaning feces out of containers every other day until they undergo the third larval molt (usually 6-9 days after hatching). This is the 4^th instar larval stage, characterized by the appearance of black hook-like crochets on each proleg and increased prominence of stripes along the insect body (Figure 1D). The larvae may vary considerably in size, but typically weigh 0.15-0.4 g upon entering the 4^th instar stage. Larvae of similar size should be chosen for injection.

2. Preparation of Bacteria for Injection

1. Grow the bacterial strain(s) to be tested according to standard procedures to the growth phase you wish to test.
2. Pellet 500 μl of each bacterial strain to be tested by spinning for 2 min in a microcentrifuge at 13,000 rpm at room temperature.
3. Resuspend cells in 1 ml sterile 1x phosphate buffered saline (PBS) and pellet again as above.
4. Resuspend cells in 0.5 ml sterile 1x PBS and measure the optical density (OD) of the suspension. Dilute all suspensions to the same OD, if necessary. Sterile growth media may also be used for cell resuspension and dilution in place of 1x PBS, if desired.

3. Injection of 4^th Instar Larvae

1. Prepare serial 10-fold dilutions of the first bacterial strain in 6 wells of a 96-well microtiter plate in sterile 1x PBS, using a fresh pipette tip for each dilution (Figure 2A). The final volume of cell suspension in each well should exceed the volume required for injection (10 μl per insect) plus 20 μl to plate the inoculum (see steps 3.2 and 3.7).
2. Spot 10 μl each for 5 dilutions (10^-2 through 10^-6) along the top of a Petri plate containing an appropriate growth medium. Tilt the plate so that the spots spread to the center (Figure 2B).
3. Remove insect diet and feces and place insects in containers on ice for approximately 5 min.
4. Sterilize the syringe needle by rinsing 3 times each in 2 tubes of 70-100% ethanol, followed by one tube of sterile H₂O (Figure 2C). The volume of liquid must be sufficient to submerge the needle.
5. Quantitate the dilute cell suspensions under a microscope using a hemocytometer. Depending on the desired inoculum, draw 10 μl from the appropriate microtiter well (Figure 2D).
6. Swab the insect surface with 95% ethanol, and inject the 10 ml cell suspension into the insect at an angle (less than 45°) behind the one of the abdominal prolegs. Take care not to puncture the gut and inject the contents just underneath the epidermal layer (Figure 2E). While some loss of hemolymph (clear, green-blue liquid) is normal, particulate matter and yellow liquid emerging from the injection site are indicative of gut puncture. If this occurs, sacrifice the insect and obtain a new larva to replace it.
7. Repeat step 3.6 for each remaining insect in the first injection group. Needle sterilization is not necessary between each insect injected with the same dilution and strain of bacteria unless contamination occurs. Alternatively, a repeating dispenser may be used for greater consistency in injection volume among samples.
8. After injecting each insect with the first strain of bacteria, repeat step 3.2, this time spotting cell suspensions in the middle of the plate and letting them flow toward the bottom (Figure 2F). Incubate plates at appropriate temperature and count colonies to enumerate the inoculum. This second plating step is used to verify that the samples were not contaminated during the injection process, as this would result in disparate colony numbers between the first and second platings.
9. Repeat steps 3.1 through 3.8 for each strain of bacteria in the experiment. Finally, inject 3-5 insects with sterile 1x PBS using a sterile needle as a negative control group. Incubate insects at 26 °C with a 16 hr light: 8 hr dark photoperiod.
10. Monitor insect survival over time after injection. Insect death is characterized as a lack of voluntary movement upon stimulation. Insects often exhibit appetite loss, diarrhea (watery, yellow excrement), and/or water loss (“sweating”) during infection prior to death; these characteristics may be worth noting as well.