We would like to thank Elisabeth Guillemet, Christophe Buisson and Ludovic Bridoux for excellent technical assistance. We are greatly indebted to Sylvie Salamitou and Sinda Fedhila for the initial system setup.

1. Insect Rearing
The whole cycle from egg to last instar larvae lasts about 5 weeks at 25 &deg;C. One or 2 additional weeks are needed to obtain adult butterflies. 
<ol>
 <li>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.</li>
 <l...

<ol>
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Immunol. 25, 697-743 (2007).</li><li>Vodovar, N., Acosta, C., Lemaitre, B., Boccard, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Drosophila:+a+polyvalent+model+to+decipher+host-pathogen+interactions.">Drosophila: a polyvalent model to decipher host-pathogen interactions.</a> Trends Microbiol. 12, 235-242 (2004).</li><li>Dalhammar, G., Steiner, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+inhibitor+A,+a+protease+from+Bacillus+thuringiensis+which+degrades+attacins+and+cecropins,+two+classes+of+antibacterial+proteins+in+insects.">Characterization of inhibitor A, a protease from Bacillus thuringiensis which degrades attacins and cecropins, two classes of antibacterial proteins in insects.</a> Eur. J. Biochem. 139, 247-252 (1984).</li><li>Jander, G., Rahme, L. G., Ausubel, F. 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S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Serological+responses+of+insects.">Serological responses of insects.</a> Fed. Proc. 26, 1675-1679 (1967).</li><li>Chadwick, J. S., Caldwell, S. S., Chadwick, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adherence+patterns+and+virulence+for+Galleria+mellonella+larvae+of+isolates+of+Serratia+marcescens.">Adherence patterns and virulence for Galleria mellonella larvae of isolates of Serratia marcescens.</a> J. Invertebr. Pathol. 55, 133-134 (1990).</li><li>Gao, W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Two+novel+point+mutations+in+clinical+Staphylococcus+aureus+reduce+linezolid+susceptibility+and+switch+on+the+stringent+response+to+promote+persistent+infection.">Two novel point mutations in clinical Staphylococcus aureus reduce linezolid susceptibility and switch on the stringent response to promote persistent infection.</a> PLoS Pathog. 6, e1000944 (2010).</li><li>Peleg, A. Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reduced+susceptibility+to+vancomycin+influences+pathogenicity+in+Staphylococcus+aureus+infection.">Reduced susceptibility to vancomycin influences pathogenicity in Staphylococcus aureus infection.</a> J. Infect. 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Microbiol. 48, 1358-1365 (2010).</li><li>Rejasse, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Temperature-dependent+production+of+various+PlcR-controlled+virulence+factors+in+Bacillus+weihenstephanensis+strain+KBAB4.">Temperature-dependent production of various PlcR-controlled virulence factors in Bacillus weihenstephanensis strain KBAB4.</a> Appl. Environ. Microbiol. 78, 2553-2557 (2012).</li><li>Jones, R. T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Photorhabdus+adhesion+modification+protein+(Pam)+binds+extracellular+polysaccharide+and+alters+bacterial+attachment.">Photorhabdus adhesion modification protein (Pam) binds extracellular polysaccharide and alters bacterial attachment.</a> BMC Microbiol. 10, 141 (2010).</li><li>Finney, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Probit analysis. , (1971).</li><li>Fedhila, S., Nel, P., Lereclus, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+InhA2+metalloprotease+of+Bacillus+thuringiensis+strain+407+is+required+for+pathogenicity+in+insects+infected+via+the+oral+route.">The InhA2 metalloprotease of Bacillus thuringiensis strain 407 is required for pathogenicity in insects infected via the oral route.</a> J. Bacteriol. 184, 3296-3304 (2002).</li><li>Guillemet, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+InhA+metalloproteases+of+Bacillus+cereus+contribute+concomitantly+to+virulence.">The InhA metalloproteases of Bacillus cereus contribute concomitantly to virulence.</a> J. Bacteriol. 192, 286-294 (2010).</li><li>Nielsen-LeRoux, C., Gaudriault, S., Ramarao, N., Lereclus, D., Givaudan, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=How+the+insect+pathogen+bacteria+Bacillus+thuringiensis+and+Xenorhabdus/Photorhabdus+occupy+their+hosts.">How the insect pathogen bacteria Bacillus thuringiensis and Xenorhabdus/Photorhabdus occupy their hosts.</a> Curr. Opin. Microbiol. 15, 1-12 (2012).</li><li>Bottone, E. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bacillus+cereus,+a+volatile+human+pathogen.">Bacillus cereus, a volatile human pathogen.</a> Clin. Microbiol. Rev. 23, 382-398 (2010).</li><li>Stenfors Arnesen, L., Fagerlund, A., Granum, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=From+soil+to+gut:+Bacillus+cereus+and+its+food+poisoning+toxins.">From soil to gut: Bacillus cereus and its food poisoning toxins.</a> FEMS Microbiol. Rev. 32, 579-606 (2008).</li><li>Lecadet, M., Blondel, M. O., Ribier, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generalized+transduction+in+Bacillus+thuringiensis+var.+berliner+1715,+using+bacteriophage+CP54.">Generalized transduction in Bacillus thuringiensis var. berliner 1715, using bacteriophage CP54.</a> Ber. J. Gen. Microbiol. 121, 203-212 (1980).</li><li>Sanchis, V., Agaisse, H., Chaufaux, J., Lereclus, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Construction+of+new+insecticidal+Bacillus+thuringiensis+recombinant+strains+by+using+the+sporulation+non-dependent+expression+system+of+cryIIIA+and+a+site+specific+recombination+vector.">Construction of new insecticidal Bacillus thuringiensis recombinant strains by using the sporulation non-dependent expression system of cryIIIA and a site specific recombination vector.</a> J. Biotechnol. 48, 81-96 (1996).</li><li>Tran, S. L., Guillemet, E., Gohar, M., Lereclus, D., Ramarao, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CwpFM+(EntFM)+is+a+Bacillus+cereus+potential+cell+wall+peptidase+implicated+in+adhesion,+biofilm+formation+and+virulence.">CwpFM (EntFM) is a Bacillus cereus potential cell wall peptidase implicated in adhesion, biofilm formation and virulence.</a> J. Bacteriol. 192, 2638-2642 (2010).</li><li>Tran, S. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hemolysin+II+is+a+Bacillus+cereus+virulence+factor+that+induces+apoptosis+of+macrophages.">Hemolysin II is a Bacillus cereus virulence factor that induces apoptosis of macrophages.</a> Cell Microbiol. 13, 92-108 (2011).</li><li>Fedhila, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparative+analysis+of+the+virulence+of+invertebrate+and+mammalian+pathogenic+bacteria+in+the+oral+insect+infection+model+Galleria+mellonella.">Comparative analysis of the virulence of invertebrate and mammalian pathogenic bacteria in the oral insect infection model Galleria mellonella.</a> J. Invertebr. 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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 stage1,2. The lepidopteran insect G. mellonella has also been mainly used for assaying bacterial virulence by injection. The advantage of tolerating higher temperatures (above 37 &deg;C) than Drosophila (maximum 25 &deg;C) is important when mammal pathogens are to be studied...

<table border="1">
	<tbody>
		<tr>
			<td>Name of the reagent</td>
			<td>Company</td>
			<td>Catalogue number</td>
			<td>Comments (optional)</td>
		</tr>
		<tr>
			<td>Wax and pollen</td>
			<td>La Ruche Roanaise</td>
			<td>303000</td>
			<td>Any honey producer</td>
		</tr>
		<tr>
			<td>Automated syringe pump</td>
			<td>KD Scientific</td>
			<td>KDS 100</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Syringe 1 ml</td>
			<td>Terumo</td>
			<td>BS 01T</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Needle 0.45 x 12 mm</td>
			<td>Terumo</td>
			<td>NN 2613R</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Petri dish 5 cm</td>
			<td>VWR</td>
			<td>89000-300</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Needle 30G, 25 mm hypodermic</td>
			<td>Burkard Mfg. Co. Ltd.</td>
			<td>PDE0005</td>
			<td>&nbsp;</td>
		</tr>
	</tbody>
</table>

the insect galleria mellonella as a powerful infection model to investigate bacterial pathogenesis

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 peptides2,3. 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 faecalis4-7. 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 model8-11. 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 &deg;C to 30 &deg;C) and infection studies can be conducted between 15 &deg;C to above 37 &deg;C12,13, 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 survival15,16 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 infections18,19.

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...

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

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.

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

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 ...

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Immunology and Infection

31.5K Views.  INRA, Micalis UMR1319, France. 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.