Methods and Challenges of Using the Greater Wax Moth (Galleria mellonella) as a Model Organism in Antimicrobial Compound Discovery

Abstract

Among non-mammalian infection model organisms, the larvae of the greater wax moth Galleria mellonella have seen increasing popularity in recent years. Unlike other invertebrate models, these larvae can be incubated at 37 °C and can be dosed relatively precisely. Despite the increasing number of publications describing the use of this model organism, there is a high variability with regard to how the model is produced in different laboratories, with respect to larva size, age, origin, storage, and rest periods, as well as dosing for infection and treatment. Here, we provide suggestions regarding how some of these factors can be approached, to facilitate the comparability of studies between different laboratories. We introduce a linear regression curve correlating the total larva weight to the liquid volume in order to estimate the in vivo concentration of pathogens and the administered drug concentration. Finally, we discuss several other aspects, including in vivo antibiotic stability in larvae, the infection doses for different pathogens and suggest guidelines for larvae selection.

Keywords: Galleria mellonella; antimicrobial compound; infection model; invertebrate model.

Figure 1

G. mellonella larva total weight versus the liquid volume curve. Healthy larvae of different sizes (n = 66) were weighed and freeze-dried. The wet-minus-dry weight was the larva liquid weight, which was found to be almost identical to the liquid volume. Each triangle represents data from a single larva. The regression line was drawn, and the coefficient of determination R square was calculated in Excel.

Figure 2

Lethal and non-lethal infection doses for different sizes of larvae. Larvae of different weights were infected with a known lethal and a known non-lethal concentration of E. coli. These concentrations were obtained in vivo based on the liquid volume calculation equation presented in Figure 1. The theoretical concentrations of the lethal and non-lethal cultures were confirmed to have the correct number of bacteria based on standard CFU counts. Larvae of different weights receiving a lethal dose had 0% survival after 24 h, while the larvae receiving a non-lethal dose survived.

Figure 3

In vivo bacterial load in G. mellonella larvae. Larvae were infected with a known concentration of E. coli equal to 1.3 × 10⁷ CFU/larva, as confirmed by plate counting. The theoretical in vivo bacterial concentration was calculated based on larva liquid volume calculated from the curve. Immediately after infection, larvae were sacrificed and bacterial load was determined in their haemolymph by viable counts. The theoretically calculated (○) in vivo concentration was very similar to the measured (∆) concentration.

Figure 4

Pathogenicity of different bacterial species in G. mellonella larvae at 24 h post-infection. Larvae (n = 5–6) were infected with decreasing concentrations of each of the following pathogens: E. coli, S. aureus, methicillin–resistant S. aureus, S. epidermidis, and P. aeruginosa. Larvae injected with PBS served as a control and showed 100% survival (bottom right corner). The bacterial inoculum (CFU/larva), for each infection group, is presented on top of each picture. The survival (%) of the different infection groups 24 h post-infection is presented in the bottom right part of each picture.

Figure 5

Example of the calculation of the concentration of the injected compound solution. In cases where a specific in vivo concentration is required (C_{in vivo}), the concentration of the injected compound solution (C_compound) can be calculated, for a specific volume of injection (V_compound, typically 10 µL) and for any larva volume (V_larva). The V_larva is the sum of the larva liquid volume (V_liquid), as calculated from the curve presented in Figure 1 and the injection volumes (typically two injections, one for the infection V_infection and one for the treatment, 10 µL each).

Figure 6

Antibiotic stability in vivo in G. mellonella larvae haemolymph. Ciprofloxacin (cip) at 490 µg/mL was injected in healthy larvae and after 10 min, 1, 3, and 24 h; the haemolymph was collected from one larva. Drops of 10 µL haemolymph were plated on agar and left to dry, after which a loan of E. coli was streaked on top. Inhibition zones were observed the next day. Larvae injected with PBS served as a control. Inhibition zones formed around the haemolymph spot from the antibiotic-injected larvae at 10 min, 1 and 3 h post-injection, but were not present at 24 h. The control haemolymph from the PBS-injected larvae did not form inhibition zones.