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

Summary

Oral and intra haemocolic infection of larvae of the greater wax moth Galleria mellonella is described. This insect can be used to study virulence factors of entomopathogenic as well as mammalian opportunistic bacteria. Rearing of the insects, methods of infection and examples of in vivo analysis are described.

Abstract

The study of bacterial virulence often requires a suitable animal model. Mammalian models of infection are costly and may raise ethical issues. The use of insects as infection models provides a valuable alternative. Compared to other non-vertebrate model hosts such as nematodes, insects have a relatively advanced system of antimicrobial defenses and are thus more likely to produce information relevant to the mammalian infection process. Like mammals, insects possess a complex innate immune system¹. Cells in the hemolymph are capable of phagocytosing or encapsulating microbial invaders, and humoral responses include the inducible production of lysozyme and small antibacterial peptides^2,3. In addition, analogies are found between the epithelial cells of insect larval midguts and intestinal cells of mammalian digestive systems. Finally, several basic components essential for the bacterial infection process such as cell adhesion, resistance to antimicrobial peptides, tissue degradation and adaptation to oxidative stress are likely to be important in both insects and mammals¹. Thus, insects are polyvalent tools for the identification and characterization of microbial virulence factors involved in mammalian infections.

Larvae of the greater wax moth Galleria mellonella have been shown to provide a useful insight into the pathogenesis of a wide range of microbial infections including mammalian fungal (Fusarium oxysporum, Aspergillus fumigatus, Candida albicans) and bacterial pathogens, such as Staphylococcus aureus, Proteus vulgaris, Serratia marcescens Pseudomonas aeruginosa, Listeria monocytogenes or Enterococcus faecalis^4-7. Regardless of the bacterial species, results obtained with Galleria larvae infected by direct injection through the cuticle consistently correlate with those of similar mammalian studies: bacterial strains that are attenuated in mammalian models demonstrate lower virulence in Galleria, and strains causing severe human infections are also highly virulent in the Galleria model^8-11. Oral infection of Galleria is much less used and additional compounds, like specific toxins, are needed to reach mortality.

G. mellonella larvae present several technical advantages: they are relatively large (last instar larvae before pupation are about 2 cm long and weight 250 mg), thus enabling the injection of defined doses of bacteria; they can be reared at various temperatures (20 °C to 30 °C) and infection studies can be conducted between 15 °C to above 37 °C^12,13, allowing experiments that mimic a mammalian environment. In addition, insect rearing is easy and relatively cheap. Infection of the larvae allows monitoring bacterial virulence by several means, including calculation of LD₅₀¹⁴, measurement of bacterial survival^15,16 and examination of the infection process¹⁷. Here, we describe the rearing of the insects, covering all life stages of G. mellonella. We provide a detailed protocol of infection by two routes of inoculation: oral and intra haemocoelic. The bacterial model used in this protocol is Bacillus cereus, a Gram positive pathogen implicated in gastrointestinal as well as in other severe local or systemic opportunistic infections^18,19.

Protocol

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.

Results

Intra haemocoelic injection of bacteria into G. mellonella has been proven very useful for the identification of many virulence factors dealing with tissue damage and resistance to innate immune factors of several human pathogens. As an example, figure 2A represents insect mortality after injection of various doses of B. cereus bacteria (wild type and mutant strains)²². Figure 2B represents bacterial survival after infection of G. mellonella by P.......

Discussion

The use of insects and especially the larval stage, as infection models for several pathogens, is becoming frequent. A model of choice for some aspects is Drosophila (the fly model) used as both adults and larval stage^1,2. The lepidopteran insect G. mellonella has also been mainly used for assaying bacterial virulence by injection. The advantage of tolerating higher temperatures (above 37 °C) than Drosophila (maximum 25 °C) is important when mammal pathogens are to be studied.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Wax and pollen	La Ruche Roanaise	303000	Any honey producer
Automated syringe pump	KD Scientific	KDS 100
Syringe 1 ml	Terumo	BS 01T
Needle 0.45 x 12 mm	Terumo	NN 2613R
Petri dish 5 cm	VWR	89000-300
Needle 30G, 25 mm hypodermic	Burkard Mfg. Co. Ltd.	PDE0005