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>
 <li>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. </li>
 <li>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. </li>
 <li>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. </li>
 <li>	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. </li>
 <li>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. </li>
</ol>
2. Insect Selection for Infection
<ol>
 <li>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. </li>
 <li>Remove the nascent silk cocoon around the larvae. </li>
</ol>
3. Bacterial Preparation
<ol>
 <li>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 3x106 cfu/ml to 1x108 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 &deg;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 &mu;l of bacterial suspension at the desired concentration. </li>
 <li>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 &deg;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 &deg;C to remove remaining vegetative bacteria. Numerate the suspension by plating serial dilutions on LB agar plates. Each larva will receive 10 &mu;l of spore suspension at the desired concentration. </li>
</ol>
4. Cry Toxin Preparation
<ol>
 <li>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 &deg;C for 72 hr with antibiotic (erythromycin 10mg/ml) to allow full sporulation, toxin crystal production and liberation in the culture supernatant.</li>
 <li>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 &deg;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. </li>
 <li>Evaluate toxin protein concentration by classic Bradford staining on crystal solution presolubilized in 50 mM NaOH. Toxin crystals can be stored at -20 &deg;C until use. </li>
</ol>
5. Injector Preparation
<ol>
 <li>To control the precise injection volume, use an automated syringe pump (e.g. KD Scientific KDS 100) with setup speed to 1 &mu;l/sec (Figure 1A). </li>
 <li>Fill a 1 ml hypodermic syringe with 300 &mu;l 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.</li>
 <li>Place the syringe in the injector.</li>
 <li>Perform a blank injection of 10 &mu;l in an empty tube to control the injected volume. </li>
</ol>
6. Insect Intrahaemocoelic Injection
<ol>
 <li>Place the insect manually between the thumb and forefinger. Then insert the needle fixed in the injector in the larva skin (cuticle) (Figure 1C).</li>
 <li>Keep the insect in place while injecting the 10 &mu;l bacterial solution (spore or vegetative bacteria). </li>
 <li>Carefully remove the insect from the needle.</li>
 <li>Place the infected insect in a small Petri dish (5 cm in diameter), 5 larvae per dish. </li>
 <li>Infect at least 20 larvae for each experimental condition.</li>
 <li>Place the dishes at 37 &deg;C in an incubator for 48 hr. </li>
</ol>
7. Insect Force Feeding (Ingestion)
<ol>
 <li>In a 2 ml Eppendorf tube, mix 0.2 &mu;g/&mu;l of Cry1C toxin with either spore or vegetative cell suspension to obtain final concentrations ranging from 3x104 to 1x107 per 10 &mu;l.</li>
 <li>Fill a 1 ml syringe with a 30G, 25 mm hypodermic needle with 300 &mu;l of the bacterial-Cry1C toxin solution for the infection of 20-25 larvae. Remove the bubbles from the syringe. </li>
 <li>Place the insect manually so that its mouth enters the needle fixed in the injector (Figure 1D), and force-feed it with 10 &mu;l of the bacterial solution using the automated syringe pump. </li>
 <li>Infect at least 20 larvae for each experimental condition. </li>
 <li>Place the infected insect in a small 5 cm Petri dish (5 larvae per dish). Place the dishes at 37 &deg;C in an incubator for 48 hr. </li>
</ol>
8. Recording Insect Mortality
<ol>
 <li>After force-feeding or injection experiments, keep the larvae in the Petri dish without food at 25 &deg;C or 37 &deg;C. Check larval mortality regularly over a 48 hr period. Dead larvae are inert and usually turn black (Figure 1B). </li>
 <li>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. </li>
</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. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Positive+correlation+between+virulence+of+Pseudomonas+aeruginosa+mutants+in+mice+and+insects.">Positive correlation between virulence of Pseudomonas aeruginosa mutants in mice and insects.</a> J. Bacteriol. 182, 3843-3845 (2000).</li><li>Purves, J., Cockayne, A., Moody, P. C., Morrissey, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+the+regulation,+metabolic+functions,+and+roles+in+virulence+of+the+glyceraldehyde-3-phosphate+dehydrogenase+homologues+gapA+and+gapB+in+Staphylococcus+aureus.">Comparison of the regulation, metabolic functions, and roles in virulence of the glyceraldehyde-3-phosphate dehydrogenase homologues gapA and gapB in Staphylococcus aureus.</a> Infect. Immun. 78, 5223-5232 (2010).</li><li>Chadwick, J. 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. Dis. 199, 532-536 (2009).</li><li>Salamitou, 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=The+plcR+regulon+is+involved+in+the+opportunistic+properties+of+Bacillus+thuringiensis+and+Bacillus+cereus+in+mice+and+insects.">The plcR regulon is involved in the opportunistic properties of Bacillus thuringiensis and Bacillus cereus in mice and insects.</a> Microbiology. 146, 2825-2832 (2000).</li><li>Cadot, C., 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=InhA1,+NprA+and+HlyII+as+candidates+to+differentiate+pathogenic+from+non-pathogenic+Bacillus+cereus+strains.">InhA1, NprA and HlyII as candidates to differentiate pathogenic from non-pathogenic Bacillus cereus strains.</a> J. Clin. 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. Pathol. 103, 24-29 (2010).</li><li>Daou, N., 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=IlsA,+a+unique+surface+protein+of+Bacillus+cereus+required+for+iron+acquisition+from+heme,+hemoglobin+and+ferritin.">IlsA, a unique surface protein of Bacillus cereus required for iron acquisition from heme, hemoglobin and ferritin.</a> PLoS Pathog. 5, e1000675 (2009).</li><li>Mason, K. 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=From+commensal+to+pathogen:+translocation+of+Enterococcus+faecalis+from+the+midgut+to+the+hemocoel+of+Manduca+sexta.">From commensal to pathogen: translocation of Enterococcus faecalis from the midgut to the hemocoel of Manduca sexta.</a> MBio. 2, e00065-00011 (2011).</li><li>Goldsmith, M. 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No conflicts of interest declared.

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

31.6K 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.

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

In vitro Biofilm Formation in an 8-well Chamber Slide

The chronic nature of many diseases is attributed to the formation of bacterial biofilms which are recalcitrant to traditional antibiotic therapy. Biofilms are community-associated bacteria attached to a surface and encased in a matrix. The role of the extracellular matrix is multifaceted, including facilitating nutrient acquisition, and offers significant protection against environmental stresses (e.g. host immune responses). In an effort to acquire a better understanding as to how the bacteria within a biofilm respond to environmental stresses we have used a protocol wherein we visualize bacterial biofilms which have formed in an 8-well chamber slide. The biofilms were stained with the BacLight Live/Dead stain and examined using a confocal microscope to characterize the relative biofilm size, and structure under varying incubation conditions. Z-stack images were collected via confocal microscopy and analyzed by COMSTAT. This protocol can be used to help elucidate the mechanism and kinetics by which biofilms form, as well as identify components that are important to biofilm structure and stability.

In vitro Biofilm Formation in an 8-well Chamber Slide

This article describes the procedure for the formation and visualization of a bacterial biofilm grown within an 8-well chamber slide

The chronic nature of many diseases is attributed to the formation of bacterial biofilms which are recalcitrant to traditional antibiotic therapy. ...

Using Luciferase to Image Bacterial Infections in Mice

Imaging is a valuable technique that can be used to monitor biological processes. In particular, the presence of cancer cells, stem cells, specific immune cell types, viral pathogens, parasites and bacteria can be followed in real-time within living animals 1-2. Application of bioluminescence imaging to the study of pathogens has advantages as compared to conventional strategies for analysis of infections in animal models3-4. Infections can be visualized within individual animals over time, without requiring euthanasia to determine the location and quantity of the pathogen. Optical imaging allows comprehensive examination of all tissues and organs, rather than sampling of sites previously known to be infected. In addition, the accuracy of inoculation into specific tissues can be directly determined prior to carrying forward animals that were unsuccessfully inoculated throughout the entire experiment. Variability between animals can be controlled for, since imaging allows each animal to be followed individually. Imaging has the potential to greatly reduce animal numbers needed because of the ability to obtain data from numerous time points without having to sample tissues to determine pathogen load3-4.
This protocol describes methods to visualize infections in live animals using bioluminescence imaging for recombinant strains of bacteria expressing luciferase. The click beetle (CBRLuc) and firefly luciferases (FFluc) utilize luciferin as a substrate5-6. The light produced by both CBRluc and FFluc has a broad wavelength from 500 nm to 700 nm, making these luciferases excellent reporters for the optical imaging in living animal models7-9. This is primarily because wavelengths of light greater than 600 nm are required to avoid absorption by hemoglobin and, thus, travel through mammalian tissue efficiently. Luciferase is genetically introduced into the bacteria to produce light signal10. Mice are pulmonary inoculated with bioluminescent bacteria intratracheally to allow monitoring of infections in real time. After luciferin injection, images are acquired using the IVIS Imaging System. During imaging, mice are anesthetized with isoflurane using an XGI-8 Gas Anethesia System. Images can be analyzed to localize and quantify the signal source, which represents the bacterial infection site(s) and number, respectively. After imaging, CFU determination is carried out on homogenized tissue to confirm the presence of bacteria. Several doses of bacteria are used to correlate bacterial numbers with luminescence. Imaging can be applied to study of pathogenesis and evaluation of the efficacy of antibacterial compounds and vaccines.

Methods for bioluminescence imaging of bacterial infections in living animals are decribed. Pathogens are modified to express luciferase allowing optical whole body imaging of infections in live animals. Animal models can be infected with luciferase expressing pathogens and the resulting course of disease visualized in real-time by bioluminescence imaging.

Imaging is a valuable technique that can be used to monitor biological processes.  In particular, the presence of cancer cells, stem cells, specific ...

Introducing Shear Stress in the Study of Bacterial Adhesion

During bacterial infections a sequence of interactions occur between the pathogen and its host. Bacterial adhesion to the host cell surface is often the initial and determining step of the pathogenesis. Although experimentally adhesion is mostly studied in static conditions adhesion actually takes place in the presence of flowing liquid. First encounters between bacteria and their host often occur at the mucosal level, mouth, lung, gut, eye, etc. where mucus flows along the surface of epithelial cells. Later in infection, pathogens occasionally access the blood circulation causing life-threatening illnesses such as septicemia, sepsis and meningitis. A defining feature of these infections is the ability of these pathogens to interact with endothelial cells in presence of circulating blood. The presence of flowing liquid, mucus or blood for instance, determines adhesion because it generates a mechanical force on the pathogen. To characterize the effect of flowing liquid one usually refers to the notion of shear stress, which is the tangential force exerted per unit area by a fluid moving near a stationary wall, expressed in dynes/cm2. Intensities of shear stress vary widely according to the different vessels type, size, organ, location etc. (0-100 dynes/cm2). Circulation in capillaries can reach very low shear stress values and even temporarily stop during periods ranging between a few seconds to several minutes 1. On the other end of the spectrum shear stress in arterioles can reach 100 dynes/cm2 2. The impact of shear stress on different biological processes has been clearly demonstrated as for instance during the interaction of leukocytes with the endothelium 3. To take into account this mechanical parameter in the process of bacterial adhesion we took advantage of an experimental procedure based on the use of a disposable flow chamber 4. Host cells are grown in the flow chamber and fluorescent bacteria are introduced in the flow controlled by a syringe pump. We initially focused our investigations on the bacterial pathogen Neisseria meningitidis, a Gram-negative bacterium responsible for septicemia and meningitis. The procedure described here allowed us to study the impact of shear stress on the ability of the bacteria to: adhere to cells 1, to proliferate on the cell surface 5and to detach to colonize new sites 6 (Figure 1). Complementary technical information can be found in reference 7. Shear stress values presented here were chosen based on our previous experience1 and to represent values found in the literature. The protocol should be applicable to a wide range of pathogens with specific adjustments depending on the objectives of the study.

During the infection process, a key step is the adhesion of pathogens with host cells. In most instances this adhesion step occurs in the presence of mechanical stress generated by flowing liquid. We describe a technique that introduces shear stress as an important parameter in the study of bacterial adhesion.

During bacterial infections a sequence of interactions occur between the pathogen and its host. Bacterial adhesion to the host cell surface is often the ...

Saliva, Salivary Gland, and Hemolymph Collection from Ixodes scapularis Ticks

Ticks are found worldwide and afflict humans with many tick-borne illnesses. Ticks are vectors for pathogens that cause Lyme disease and tick-borne relapsing fever (Borrelia spp.), Rocky Mountain Spotted fever (Rickettsia rickettsii), ehrlichiosis (Ehrlichia chaffeensis and E. equi), anaplasmosis (Anaplasma phagocytophilum), encephalitis (tick-borne encephalitis virus), babesiosis (Babesia spp.), Colorado tick fever (Coltivirus), and tularemia (Francisella tularensis) 1-8. To be properly transmitted into the host these infectious agents differentially regulate gene expression, interact with tick proteins, and migrate through the tick 3,9-13. For example, the Lyme disease agent, Borrelia burgdorferi, adapts through differential gene expression to the feast and famine stages of the tick's enzootic cycle 14,15. Furthermore, as an Ixodes tick consumes a bloodmeal Borrelia replicate and migrate from the midgut into the hemocoel, where they travel to the salivary glands and are transmitted into the host with the expelled saliva 9,16-19.
As a tick feeds the host typically responds with a strong hemostatic and innate immune response 11,13,20-22. Despite these host responses, I. scapularis can feed for several days because tick saliva contains proteins that are immunomodulatory, lytic agents, anticoagulants, and fibrinolysins to aid the tick feeding 3,11,20,21,23. The immunomodulatory activities possessed by tick saliva or salivary gland extract (SGE) facilitate transmission, proliferation, and dissemination of numerous tick-borne pathogens 3,20,24-27. To further understand how tick-borne infectious agents cause disease it is essential to dissect actively feeding ticks and collect tick saliva. This video protocol demonstrates dissection techniques for the collection of hemolymph and the removal of salivary glands from actively feeding I. scapularis nymphs after 48 and 72 hours post mouse placement. We also demonstrate saliva collection from an adult female I. scapularis tick.

Saliva, Salivary Gland, and Hemolymph Collection from Ixodes scapularis Ticks

The collection of infected tick hemolymph, salivary glands, and saliva is important to study how tick-borne pathogens cause disease. In this protocol we demonstrate how to collect hemolymph and salivary glands from feeding Ixodes scapularis nymphs. We also demonstrate saliva collection from female I. scapularis adults.

Ticks are found worldwide and afflict humans with many tick-borne illnesses. Ticks are vectors for pathogens that cause Lyme disease and tick-borne ...

A Visual Assay to Monitor T6SS-mediated Bacterial Competition

Type VI secretion systems (T6SSs) are molecular nanomachines allowing Gram-negative bacteria to transport and inject proteins into a wide variety of target cells1,2. The T6SS is composed of 13 core components and displays structural similarities with the tail-tube of bacteriophages3. The phage uses a tube and a puncturing device to penetrate the cell envelope of target bacteria and inject DNA. It is proposed that the T6SS is an inverted bacteriophage device creating a specific path in the bacterial cell envelope to drive effectors and toxins to the surface. The process could be taken further and the T6SS device could perforate other cells with which the bacterium is in contact, thus injecting the effectors into these targets. The tail tube and puncturing device parts of the T6SS are made with Hcp and VgrG proteins, respectively4,5.
The versatility of the T6SS has been demonstrated through studies using various bacterial pathogens. The Vibrio cholerae T6SS can remodel the cytoskeleton of eukaryotic host cells by injecting an "evolved" VgrG carrying a C-terminal actin cross-linking domain6,7. Another striking example was recently documented using Pseudomonas aeruginosa which is able to target and kill bacteria in a T6SS-dependent manner, therefore promoting the establishment of bacteria in specific microbial niches and competitive environment8,9,10.
In the latter case, three T6SS-secreted proteins, namely Tse1, Tse2 and Tse3 have been identified as the toxins injected in the target bacteria (Figure 1). The donor cell is protected from the deleterious effect of these effectors via an anti-toxin mechanism, mediated by the Tsi1, Tsi2 and Tsi3 immunity proteins8,9,10. This antimicrobial activity can be monitored when T6SS-proficient bacteria are co-cultivated on solid surfaces in competition with other bacterial species or with T6SS-inactive bacteria of the same species8,11,12,13.
The data available emphasized a numerical approach to the bacterial competition assay, including time-consuming CFU counting that depends greatly on antibiotic makers. In the case of antibiotic resistant strains like P. aeruginosa, these methods can be inappropriate. Moreover, with the identification of about 200 different T6SS loci in more than 100 bacterial genomes14, a convenient screening tool is highly desirable. We developed an assay that is easy to use and requires standard laboratory material and reagents. The method offers a rapid and qualitative technique to monitor the T6SS-dependent bactericidal/bacteriostasis activity by using a reporter strain as a prey (in this case Escherichia coli DH5&alpha;) allowing a-complementation of the lacZ gene. Overall, this method is graphic and allows rapid identification of T6SS-related phenotypes on agar plates. This experimental protocol may be adapted to other strains or bacterial species taking into account specific conditions such as growth media, temperature or time of contact.

We describe a qualitative assay to monitor bacterial competition mediated by the Pseudomonas aeruginosa type VI secretion system (T6SS). The assay relies on the survival/killing of Escherichia coli target cells carrying a lacZ-reporter. This technique is adjustable to assess the bactericidal/bacteriostasis activity of T6SS-proficient microorganisms.

Type VI secretion systems (T6SSs) are molecular  nanomachines allowing Gram-negative bacteria to transport and inject proteins  into a wide variety of ...

Use of Galleria mellonella as a Model Organism to Study Legionella pneumophila Infection

Legionella pneumophila, the causative agent of a severe pneumonia named Legionnaires&#39; disease, is an important human pathogen that infects and replicates within alveolar macrophages. Its virulence depends on the Dot/Icm type IV secretion system (T4SS), which is essential to establish a replication permissive vacuole known as the Legionella containing vacuole (LCV). L. pneumophila infection can be modeled in mice however most mouse strains are not permissive, leading to the search for novel infection models. We have recently shown that the larvae of the wax moth Galleria mellonella are suitable for investigation of L. pneumophila infection. G. mellonella is increasingly used as an infection model for human pathogens and a good correlation exists between virulence of several bacterial species in the insect and in mammalian models. A key component of the larvae&#39;s immune defenses are hemocytes, professional phagocytes, which take up and destroy invaders. L. pneumophila is able to infect, form a LCV and replicate within these cells. Here we demonstrate protocols for analyzing L. pneumophila virulence in the G. mellonella model, including how to grow infectious L. pneumophila, pretreat the larvae with inhibitors, infect the larvae and how to extract infected cells for quantification and immunofluorescence microscopy. We also describe how to quantify bacterial replication and fitness in competition assays. These approaches allow for the rapid screening of mutants to determine factors important in L. pneumophila virulence, describing a new tool to aid our understanding of this complex pathogen.

Use of Galleria mellonella as a Model Organism to Study Legionella pneumophila Infection

The larva of the wax moth Galleria mellonella was recently established as an in vivo model to study Legionella pneumophila infection. Here, we demonstrate fundamental techniques to characterize the pathogenesis of Legionella in the larvae, including inoculation, measurement of bacterial virulence and replication as well as extraction and analysis of infected hemocytes.

Legionella pneumophila, the causative agent of a severe pneumonia named Legionnaires&#39; disease, is an important human pathogen that infects and ...

Imaging InlC Secretion to Investigate Cellular Infection by the Bacterial Pathogen Listeria monocytogenes

Bacterial intracellular pathogens can be conceived as molecular tools to dissect cellular signaling cascades due to their capacity to exquisitely manipulate and subvert cell functions which are required for the infection of host target tissues. Among these bacterial pathogens, Listeria monocytogenes is a Gram positive microorganism that has been used as a paradigm for intracellular parasitism in the characterization of cellular immune responses, and which has played instrumental roles in the discovery of molecular pathways controlling cytoskeletal and membrane trafficking dynamics. In this article, we describe a robust microscopical assay for the detection of late cellular infection stages of L. monocytogenes based on the fluorescent labeling of InlC, a secreted bacterial protein which accumulates in the cytoplasm of infected cells; this assay can be coupled to automated high-throughput small interfering RNA screens in order to characterize cellular signaling pathways involved in the up- or down-regulation of infection.

Imaging InlC Secretion to Investigate Cellular Infection by the Bacterial Pathogen Listeria monocytogenes

Listeria monocytogenes is a Gram positive bacterial pathogen frequently used as a major model for the study of intracellular parasitism. Imaging late L. monocytogenes infection stages within the context of small-interfering RNA screens allows for the global study of cellular pathways required for bacterial infection of target host cells.

Bacterial intracellular  pathogens can be conceived as molecular tools to dissect cellular signaling  cascades due to their capacity to exquisitely ...

Zebrafish as a Model to Assess the Teratogenic Potential of Nitrite

High nitrate levels in the environment may&#160;result in congenital defects or miscarriages in humans. Presumably, this is due to the conversion of nitrate to nitrite by gut and salivary bacteria. However, in other mammalian studies, high nitrite levels do not cause birth defects, although they can lead to poor reproductive outcomes. Thus, the teratogenic potential of nitrite is not clear. It would be useful to have a vertebrate model system to easily assess teratogenic effects of nitrite or any other chemical of interest. Here, we demonstrate the utility of zebrafish (Danio rerio) to screen compounds for toxicity and embryonic defects. Zebrafish embryos are fertilized externally and have rapid development, making them a good model for teratogenic studies. We show that increasing the time of exposure to nitrite negatively affects survival. Increasing the concentration of nitrite also adversely affects survival, whereas nitrate does not. For embryos that survive nitrite exposure, various defects can occur, including pericardial and yolk sac&#160;edema, swim bladder noninflation, and craniofacial malformation. Our results indicate that the zebrafish is a convenient system for studying the teratogenic potential of nitrite. This approach can easily be adapted to test other chemicals for their effects on early vertebrate development.

Exposure to teratogens may cause birth defects. Zebrafish are useful for determining the teratogenic potential of chemicals. We demonstrate the utility of zebrafish by exposing embryos to various levels of nitrite and also at different times of exposure. We show that nitrite can be toxic and cause severe developmental defects.

High nitrate levels in the environment may&#160;result in congenital defects or miscarriages in humans. Presumably, this is due to the conversion of ...

The Madagascar Hissing Cockroach as an Alternative Non-mammalian Animal Model to Investigate Virulence, Pathogenesis, and Drug Efficacy

Many aspects of innate immunity are conserved between mammals and insects. An insect, the Madagascar hissing cockroach from the genus Gromphadorhina, can be utilized as an alternative animal model for the study of virulence, host-pathogen interaction, innate immune response, and drug efficacy. Details for the rearing, care and breeding of the hissing cockroach are provided. We also illustrate how it can be infected with bacteria such as the intracellular pathogens Burkholderia mallei, B. pseudomallei, and B. thailandensis. Use of the hissing cockroach is inexpensive and overcomes regulatory issues dealing with the use of mammals in research. In addition, results found using the hissing cockroach model are reproducible and similar to those obtained using mammalian models. Thus, the Madagascar hissing cockroach represents an attractive surrogate host that should be explored when conducting animal studies.

We present a protocol to utilize the Madagascar hissing cockroach as an alternative non-mammalian animal model to conduct bacterial virulence, pathogenesis, drug toxicity, drug efficacy, and innate immune response studies.

Many aspects of innate immunity are conserved between mammals and insects. An insect, the Madagascar hissing cockroach from the genus Gromphadorhina, can ...

An Ex Vivo Chicken Primary Bursal-cell Culture Model to Study Infectious Bursal Disease Virus Pathogenesis

Infectious bursal disease virus (IBDV) is a birnavirus of economic importance to the poultry industry. The virus infects B cells, causing morbidity, mortality, and immunosuppression in infected birds. In this study, we describe the isolation of chicken primary bursal cells from the bursa of Fabricius, the culture and infection of the cells with IBDV, and the quantification of viral replication. The addition of chicken CD40 ligand significantly increased cell proliferation fourfold over six days of culture and significantly enhanced cell viability. Two strains of IBDV, a cell-culture adapted strain, D78, and a very virulent strain, UK661, replicated well in the ex vivo cell cultures. This model will be of use in determining how cells respond to IBDV infection and will permit a reduction in the number of infected birds used in IBDV pathogenesis studies. The model can also be expanded to include other viruses and could be applied to different species of birds.

An Ex Vivo Chicken Primary Bursal-cell Culture Model to Study Infectious Bursal Disease Virus Pathogenesis

Here we describe the isolation of chicken primary bursal cells from the bursa of Fabricius, the culture and infection of the cells with infectious bursal disease virus, and the quantification of viral replication.

Infectious bursal disease virus (IBDV) is a birnavirus of economic importance to the poultry industry. The virus infects B cells, causing morbidity, ...