From moths to caterpillars: Ideal conditions for Galleria mellonella rearing for in vivo microbiological studies

Figures & data

Figure 1. Different developmental stages of Galleria mellonella. Eggs (1), approximately 10-day-old caterpillar (2), approximately 20-day-old caterpillar (3), 25-35-day-old caterpillar (4 and 5), approximately 40-day-old caterpillar (last larval stage) (6), pre-pupae and pupae (7 and 8), adult moths (9).

Figure 2. Galleria mellonella male (A) and female (B).

Table 1. Dietary components evaluated for Galleria mellonella rearing.

Diet 1	Diet 2	Diet 3
20 g of brown sugar	300 g oat flakes	250 g of corn meal
80 g of glycerol	300 g of whole wheat flour	150 g of yeast extract
400 g of powder milk	60 g dried yeast	100 g of soy flour
120 g yeast extract	120 mL of glycerol	100 g of powder milk
200 g of whole wheat flour	120 ml of honey	200 g of honey
200 g of wheat bran	beeswax blocks	200 g of glycerol
200 g of wheat germ		beeswax blocks
beeswax blocks

Figure 3. Process for Galleria mellonella rearing.

Table 2. Larval life cycle parameters of Galleria mellonella groups fed different diets.

Diet	Larval phase (days)	Weight (g)	Pulpal (%)
Diet 1	42 ± 1.53^a	0.277 ± 0.031^a	15.2 ± 1.70^a
Diet 2	49 ± 2.52^b	0.278 ± 0.019^a	4 ± 1.29^b
Diet 3	35 ± 2.00^c	0.365 ± 0.033^b	8.4 ± 0.84^c

Mean values and standard deviations (±SD) of three independent experiments are represented. Different lowercase letters represent significant differences among the groups, according to Tukey's test (P < 0.05).

Table 3. Immune system parameters of Galleria mellonella groups fed different diets.

			Survival (%)
Diet	Hemolymph volume (µL)	Hemocyte concentrations (x10^6/mL)	S. aureus	E. coli	C. albicans
Diet 1	20 ± 4.57^a	2.4 ± 0.29^a	41.67	33.33	33.33
Diet 2	23 ± 5.29^a	2.8 ± 0.17^a	41.67	41.67	25.00
Diet 3	48 ± 4.20^b	5.7 ± 0.43^b	50.00	58.33	41.67

Mean values and standard deviations (±SD) of three independent experiments are represented. Different lowercase letters represent significant differences among the groups, according to Tukey's test (P < 0.05).

Figure 4. Survival curves of Galleria mellonella fed different diets and infected with Staphylococcus aureus (A), Escherichia coli (B) and Candida albicans (C). No significant difference was observed among the groups fed diets 1, 2 or 3 for each microorganism: S. aureus (P = 0.7367), E. coli (P = 0.4010), and C. albicans (P = 0.5027).

Table 4. Questions and tips for Galleria mellonella rearing.

Questions	Solution and tips
How many times should G. mellonella be fed?	Every two or three days is enough.
How much food should be added?	For egg/small larvae container put 5g of food and for median/large 8–10g.
How the just born larvae looks like?	Just born larvae are white and very small (around 1mm).
How to prevent very small larvae from escaping of the moth container?	Change the filter paper with the eggs more often.
How many male and female moths are necessary for egg position?	There is no need to add an exact number of male or female moths. Male moths can fertilize multiple females; moreover, the number of eggs that females lay is high.
How can the eggs be removed from the filter paper?	It is not necessary to remove the eggs from the filter paper because they are very sensitive. Transfer the paper with the eggs as described.