The Insect Galleria mellonella as a Powerful Infection Model to Investigate Bacterial Pathogenesis

1. Insect Rearing

The whole cycle from egg to last instar larvae lasts about 5 weeks at 25 °C. One or 2 additional weeks are needed to obtain adult butterflies.

1. Place at least 100 pupae or newly merged adult G. mellonella butterflies in a 5-liter wire-mesh cage. Male butterflies measure 10 to 15 mm. The adult male moth is beige with faint light and dark markings. Female butterflies measure around 20 mm. Females are darker than males with a brown/grey color.
2. Suspend two packs of four-layered paper in the cage for egg-laying. After 2 days, the adult female will lay eggs on the edge between the papers.
3. Twice a week, place the egg-paper packs in new plastic rearing boxes with grids to let air circulate, containing pollen and bee wax. Eggs hatch in about 3 days and small larvae start to develop by feeding on wax and pollen. Alternatively, larvae can be placed on an artificial diet consisting of a mix of 500 g liquid honey, 400 g glycerin, 100 g dried beer yeast, 250 g wheat flour, 200 g skim milk powder and 400 g polenta. To provide appropriate ratio of food per larva, regularly separate the larvae into new boxes and replenish the food every two days.
4. Larvae are reared during the six stages of larva stadium. Each stadium is characterized by the slippage of the head capsule of larvae. When larvae reach the last stage before pupation, they stop feeding and start building a light silk cocoon.
5. In their protective cocoons, the larvae will rest and transform into pupae. Wax worms remain in the pupa stage for one to two weeks to emerge into adult moths. The adult moths do not drink nor eat. Mating occurs and the wax worms’ life cycle begins again.
6. To standardize the pathology assay, take the larvae during the last larval stage. During this stage they stop feeding, move to the cover of the rearing box and start to produce silk. This stage lasts around 5 days.

2. Insect Selection for Infection

1. Select last instar larvae, which are 2-3 cm long and 180-250 mg in weight, 24 hr before infections and put them into an empty box to starve them.
2. Remove the nascent silk cocoon around the larvae.

3. Bacterial Preparation

1. Prepare Bacillus bacterial suspensions to obtain final concentrations ranging from 10⁴ colony forming units (cfu)/ml to 10⁸ cfu/ml for intra haemocoelic injection and from 3×10⁶ cfu/ml to 1×10⁸ cfu/ml for ingestion (the cfu to obtain a LD₅₀ depends on the bacterial species). In our case, B. cereus is grown in Luria-Bertani broth medium (LB, 10 g/l tryptone, 5 g/l yeast extract, 10 g/l NaCl) at 37 °C under agitation until entry into stationary phase, corresponding to the incurvation of the growth curve. Measure the OD 600 nm (between 1 and 2 for B. cereus) and use it to evaluate bacterial concentration, using a previously made titration curve. Harvest bacteria by centrifugation at 5,000 x g for 10 min at room temperature and resuspend in Phosphate Buffered Saline (PBS: 1M KH₂PO₄, 1M K₂HPO₄, 5M NaCl, pH 7.2). Each larva will receive 10 μl of bacterial suspension at the desired concentration.
2. Alternatively, spore suspension can be prepared for infection. Obtain spores by culturing the bacteria in the sporulation medium HCT (5 g/L tryptone, 2 g/L casein hydrolysate, 12.5 mM K₂HPO₄, 12.5 mM MgSO₄, 0.05 mM MnSO₄, 1.2 mM ZnSO₄, 1.2 mM Fe²(SO₄)³, 0.5% H₂SO₄, 25 mM CaCl₂)²⁰ at 30 °C for 3 days. Harvest spores by centrifugation (10,000 x g, 15 min), and wash twice in sterile distilled water. Resuspend the spore pellet in sterile distilled water and heat for 15 min at 78 °C to remove remaining vegetative bacteria. Numerate the suspension by plating serial dilutions on LB agar plates. Each larva will receive 10 μl of spore suspension at the desired concentration.

4. Cry Toxin Preparation

1. Prepare the Cry1C toxin from the B. thuringiensis strain 407 transformed with the plasmid pHTF31C²¹ carrying the gene encoding Cry1C. Culture the strain in 100 ml HCT medium at 30 °C for 72 hr with antibiotic (erythromycin 10mg/ml) to allow full sporulation, toxin crystal production and liberation in the culture supernatant.
2. Purify crystals from the supernatant on a 72%-79% sucrose gradient. First add 17 ml of 79% sucrose in a 40 ml tube. Carefully layer 17 ml of 72 % sucrose on top of the 79% sucrose. Finally, layer 5-6 ml of the 10X concentrated sporulated culture and close the tube. Centrifuge the tube for 14 hr at 20,000 x g, 4 °C, to separate the crystals from the spores. Collect the crystals at the gradient interface. Resuspend the crystal pellet in 25 ml cold sterile water to wash it. Centrifuge at 8,000 x g for 20 min. This step is repeated three times to remove all sucrose sticking to the crystals. Resuspend the crystals in 5 ml sterile water and observe under the microscope to evaluate purity.
3. Evaluate toxin protein concentration by classic Bradford staining on crystal solution presolubilized in 50 mM NaOH. Toxin crystals can be stored at -20 °C until use.

5. Injector Preparation

1. To control the precise injection volume, use an automated syringe pump (e.g. KD Scientific KDS 100) with setup speed to 1 μl/sec (Figure 1A).
2. Fill a 1 ml hypodermic syringe with 300 μl water or the bacterial solution (1 syringe per condition) for infection of 20-25 larvae. Remove the bubbles by carefully tapping the syringe, then attach a 0.45 x 12 mm needle to the syringe.
3. Place the syringe in the injector.
4. Perform a blank injection of 10 μl in an empty tube to control the injected volume.

6. Insect Intrahaemocoelic Injection

1. Place the insect manually between the thumb and forefinger. Then insert the needle fixed in the injector in the larva skin (cuticle) (Figure 1C).
2. Keep the insect in place while injecting the 10 μl bacterial solution (spore or vegetative bacteria).
3. Carefully remove the insect from the needle.
4. Place the infected insect in a small Petri dish (5 cm in diameter), 5 larvae per dish.
5. Infect at least 20 larvae for each experimental condition.
6. Place the dishes at 37 °C in an incubator for 48 hr.

7. Insect Force Feeding (Ingestion)

1. In a 2 ml Eppendorf tube, mix 0.2 μg/μl of Cry1C toxin with either spore or vegetative cell suspension to obtain final concentrations ranging from 3×10⁴ to 1×10⁷ per 10 μl.
2. Fill a 1 ml syringe with a 30G, 25 mm hypodermic needle with 300 μl of the bacterial-Cry1C toxin solution for the infection of 20-25 larvae. Remove the bubbles from the syringe.
3. Place the insect manually so that its mouth enters the needle fixed in the injector (Figure 1D), and force-feed it with 10 μl of the bacterial solution using the automated syringe pump.
4. Infect at least 20 larvae for each experimental condition.
5. Place the infected insect in a small 5 cm Petri dish (5 larvae per dish). Place the dishes at 37 °C in an incubator for 48 hr.

8. Recording Insect Mortality

1. After force-feeding or injection experiments, keep the larvae in the Petri dish without food at 25 °C or 37 °C. Check larval mortality regularly over a 48 hr period. Dead larvae are inert and usually turn black (Figure 1B).
2. Mortality data can be analyzed using the log-Probit program¹⁴. This program tests the linearity of dose mortality curves and provides lethal doses (LD₅₀). Alternatively, other programs, such as Prism (Graph Pad), analyzing survival or mortality data can be used.