This work was funded by the DFG (SPP1656), the DFG research training group 1708, the Bundesministerium f&#252;r Bildung und Forschung (BMBF), and the German Center for Infection Research (DZIF).

1. G. mellonella rearing and preparation of the larvae for the experiments
NOTE: The cycle from egg to last instar larva takes approximately 5-6 weeks.
<ol>
	<li>Transfer the eggs laid by adult moths to 2 L boxes containing wax moth substrate (22% corn grits, 22% wheat meal, 17.5% beeswax, 11% skimmed milk powder, 11% honey, 11% glycerol, 5.5% dried yeast). Perform the whole breeding at 30 &#176;C in the dark.</li>
	<li>Transfer 25 g of substrate containing the larvae into fresh sub...

<ol>
	<li>Nell, S., Suerbaum, S., Josenhans, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+impact+of+the+microbiota+on+the+pathogenesis+of+IBD:+lessons+from+mouse+infection+models.">The impact of the microbiota on the pathogenesis of IBD: lessons from mouse infection models.</a> Nature Reviews Microbiology. 8 (8), 564-577 (2010).</li><li>Muniz, L. R., Knosp, C., Yeretssian, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intestinal+antimicrobial+peptides+during+homeostasis,+infection,+and+disease.">Intestinal antimicrobial peptides during homeostasis, infection, and disease.</a> Frontiers in Immunology. 3, 310 (2012).</li><li>Ivanov, I. I., Honda, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intestinal+commensal+microbes+as+immune+modulators.">Intestinal commensal microbes as immune modulators.</a> Cell Host Microbe. 12 (4), 496-508 (2012).</li><li>Ayres, 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=Inflammasome-microbiota+interplay+in+host+physiologies.">Inflammasome-microbiota interplay in host physiologies.</a> Cell Host Microbe. 14 (5), 491-497 (2013).</li><li>Champion, O. L., Titball, R. W., Bates, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Standardization+of+G.+mellonella+Larvae+to+Provide+Reliable+and+Reproducible+Results+in+the+Study+of+Fungal+Pathogens.">Standardization of G. mellonella Larvae to Provide Reliable and Reproducible Results in the Study of Fungal Pathogens.</a> Journal of Fungi (Basel). 4 (3), (2018).</li><li>Wojda, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunity+of+the+greater+wax+moth+Galleria+mellonella.">Immunity of the greater wax moth Galleria mellonella.</a> Insect Science. , (2016).</li><li>Buchmann, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evolution+of+Innate+Immunity:+Clues+from+Invertebrates+via+Fish+to+Mammals.">Evolution of Innate Immunity: Clues from Invertebrates via Fish to Mammals.</a> Frontiers in Immunology. 5, 459 (2014).</li><li>Lange, 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=Galleria+mellonella:+A+Novel+Invertebrate+Model+to+Distinguish+Intestinal+Symbionts+From+Pathobionts.">Galleria mellonella: A Novel Invertebrate Model to Distinguish Intestinal Symbionts From Pathobionts.</a> Frontiers in Immunology. 9 (2114), (2018).</li><li>Tsai, C. J., Loh, J. M., Proft, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Galleria+mellonella+infection+models+for+the+study+of+bacterial+diseases+and+for+antimicrobial+drug+testing.">Galleria mellonella infection models for the study of bacterial diseases and for antimicrobial drug testing.</a> Virulence. , 1-16 (2016).</li><li>Bolouri Moghaddam, M. R., 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+potential+of+the+Galleria+mellonella+innate+immune+system+is+maximized+by+the+co-presentation+of+diverse+antimicrobial+peptides.">The potential of the Galleria mellonella innate immune system is maximized by the co-presentation of diverse antimicrobial peptides.</a> Biological Chemistry. 397 (9), 939-945 (2016).</li><li>Casanova-Torres, A. M., Goodrich-Blair, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immune+Signaling+and+Antimicrobial+Peptide+Expression+in+Lepidoptera.">Immune Signaling and Antimicrobial Peptide Expression in Lepidoptera.</a> Insects. 4 (3), 320-338 (2013).</li><li>Mukherjee, K., 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=Galleria+mellonella+as+a+model+system+for+studying+Listeria+pathogenesis.">Galleria mellonella as a model system for studying Listeria pathogenesis.</a> Applied and Environmental Microbiology. 76 (1), 310-317 (2010).</li><li>Brennan, M., Thomas, D. Y., Whiteway, M., Kavanagh, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Correlation+between+virulence+of+Candida+albicans+mutants+in+mice+and+Galleria+mellonella+larvae.">Correlation between virulence of Candida albicans mutants in mice and Galleria mellonella larvae.</a> FEMS Immunological and Medical Microbiology. 34 (2), 153-157 (2002).</li><li>Miyata, S., Casey, M., Frank, D. W., Ausubel, F. 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L., Skovgaard, K., Stockmarr, A., Larsen, N., Molbak, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Gut+Microbiotassay:+a+high-throughput+qPCR+approach+combinable+with+next+generation+sequencing+to+study+gut+microbial+diversity.">The Gut Microbiotassay: a high-throughput qPCR approach combinable with next generation sequencing to study gut microbial diversity.</a> BMC Genomics. 14, 788 (2013).</li><li>Sato, K., 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=OmpA+variants+affecting+the+adherence+of+ulcerative+colitis-derived+Bacteroides+vulgatus.">OmpA variants affecting the adherence of ulcerative colitis-derived Bacteroides vulgatus.</a> Journal of Medical and Dental Science. 57 (1), 55-64 (2010).</li><li>Freitak, D., 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+maternal+transfer+of+bacteria+can+mediate+trans-generational+immune+priming+in+insects.">The maternal transfer of bacteria can mediate trans-generational immune priming in insects.</a> Virulence. 5 (4), 547-554 (2014).</li><li>Pfaffl, M. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+mathematical+model+for+relative+quantification+in+real-time+RT-PCR.">A new mathematical model for relative quantification in real-time RT-PCR.</a> Nucleic Acids Research. 29 (9), 45 (2001).</li><li>Ramarao, N., Nielsen-Leroux, C., 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+insect+Galleria+mellonella+as+a+powerful+infection+model+to+investigate+bacterial+pathogenesis.">The insect Galleria mellonella as a powerful infection model to investigate bacterial pathogenesis.</a> Journal of Visualized Experiments. (70), e4392 (2012).</li><li>Ramarao, N., Nielsen-Leroux, C., 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+insect+Galleria+mellonella+as+a+powerful+infection+model+to+investigate+bacterial+pathogenesis.">The insect Galleria mellonella as a powerful infection model to investigate bacterial pathogenesis.</a> Journal of Visualized Experiments. (70), e4392 (2012).</li></ol>

The G. mellonella model is a frequently used model to assess bacterial virulence factors in a systemic infection approach21. Since many pathogens and bacteria enter the host via the oral colonization or infection route, new insights need to be found to evaluate G. mellonella as a model for oral colonization and infection.
The possibility to rear G. mellonella between 15-37 &#176;C is a great advantage since most mammalian models maintain body ...

The intestinal microbiome is an essential component for maintenance of homeostasis, and involves both innate and adaptive immune responses1,2. The commensal microbiota community is characterized by different main commensal constituents: symbionts that confer beneficial effects by important immunomodulatory functions, and pathobionts that can have detrimental effects in genetically predisposed hosts and promote and trigger intestinal inflammation3,4. Many studies on symbionts and pathobionts and their influence on the host immune system have been published mainly studying adaptive immune responses.
Since these studies involve many animals for the investigations and the protection and replacement of animals used for experimentation is of increasing public interest, we seek to find a replacement model to allow for a screening of different bacterial immunogenic properties. Insects, especially Galleria mellonella, are a widely used replacement model in infection research. G. mellonella combines different advantages such as low costs and high throughput; it allows oral administration of bacteria, which is the natural exposure route, and it allows for systemic infection5,6. G. mellonella further enables incubation at 37 &#176;C, which is the physiological body temperature of mammals and the optimum for bacterial virulence factor expression5. The main advantage of G. mellonella is the conserved innate immune system that enables the discrimination of self from non-self and encodes a variety of pattern recognition receptors like apolipophorin or the opsonin hemolin6,7. Upon microbe recognition, G. mellonella can trigger different downstream humoral immune responses. It can induce oxidative stress responses and secrete reactive oxygen species (ROS) which involves the activity of NOS (nitric oxidase synthase) and NOX (NADPH oxidase)6,8. In addition, G. mellonella activates a potent antimicrobial peptide (AMP) response, which results in the secretion of a mixture of different AMPs such as gloverin, moricin, cecropin or the defensin-like gallerimycin6,8,9,10. Generally, AMPs have quite broad host specificity against Gram-positive and Gram-negative bacteria and fungi and have to provide an potent response since insects are lacking any adaptive response10. Gloverin is an AMP active against bacteria and fungi and inhibits outer membrane formation6,11. Moricins exhibit their antimicrobial function against Gram-positive and Gram-negative bacteria by penetrating the membrane and forming a pore9,11. Cecropins provide activity against bacteria and fungi and permeabilize the membrane similarly like moricins9,10. Gallerimycin is a defensin-like peptide with anti-fungal properties9. Interestingly, it was found that the combination of cecropin and gallerimycin had a synergistic activity against E. coli10.
Due to their easy-to-use character G. mellonella larvae are an often used infection model to assess bacterial pathogenicity. In particular, studies in which data obtained from G. mellonella correlate with data obtained from mice support the strength of this alternative host model. It was found that the most pathogenic serotypes of Listeria monocytogenes in a mouse infection model lead also to higher mortality rates in G. mellonella after systemic infection. Further, less virulent serotypes turned out to be also less virulent in the G. mellonella model12. Similar observations have been made with the human pathogenic fungi Candida albicans. Virulence of different C. albicans strains has been assessed by systemic infection and subsequent monitoring of larval survival. Mouse avirulent strains were also avirulent or exhibited reduced virulence in G. mellonella, whereas the mouse virulent strains lead also to high larval mortality13. The G. mellonella model could further be used to identify type 3 secretion system pathogenicity factors of Pseudomonas aeruginosa14.
Since most investigations involving G. mellonella were focused on virulence factors using the systemic infection approach we were especially interested in providing a method suitable for the analysis of intestinal commensals in an oral force-feeding model in which we can apply a distinct dosage of bacteria per larvae and not only observe the larval mortality rate but analyze different hallmarks of innate immune responses to maintain intestinal homeostasis.
Our method helps to increase the use of G. mellonella as a replacement model since we combine the application of bacteria and the analysis of RNA expression. It is not only useful to strengthen the meaning of bacterial pathogenesis studies when including the analysis of immune responses after oral administration and not only the observation of mortality rates after systemic infection. Our methods allows for the analysis of immunogenic properties of bacterial non-pathogenic commensals since it is provides more complex conditions than cell culture by offering an intestinal barrier in a living organism.

<table border="0" cellpadding="0" cellspacing="0" style="width:701px;" width="702">
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		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">1.5 mL tubes</td>
			<td style="width:200px;">Eppendorf</td>
			<td style="width:119px;">0030120086</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">100 bp DNA ladder&#160;</td>
			<td style="width:200px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">15628019</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">1-Bromo-3-Chloropropane (BCP)</td>
			<td>Sigma-Aldrich</td>
			<td style="width:119px;">B9673</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">2 mL tubes</td>
			<td style="width:200px;">Eppendorf</td>
			<td style="width:119px;">0030120094</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">2x Mangomix</td>
			<td style="width:200px;">Bioline</td>
			<td style="width:119px;">BIO-25033</td>
			<td>Colony PCR</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">50 mL tubes</td>
			<td style="width:200px;">Greiner Bio-One</td>
			<td style="width:119px;">210 261</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Agarose</td>
			<td style="width:200px;">Biozym</td>
			<td style="width:119px;">840004</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Beeswax</td>
			<td style="width:200px;">Mixed-Store.de</td>
			<td style="width:119px;">&#160;-</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Brain heart infusion broth</td>
			<td>Thermo Fisher Scientific</td>
			<td style="width:119px;">CM1135</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">CloneJET PCR Cloning Kit</td>
			<td style="width:200px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">K1232</td>
			<td>Cloning vector for 16S fragments</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Corn grits</td>
			<td style="width:200px;">Osterm&#252;hle Naturkost GmbH</td>
			<td style="width:119px;">306</td>
			<td>Organic cultivation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Difco LB Agar, Miller (Luria-Bertani)</td>
			<td>Becton Dickinson</td>
			<td style="width:119px;">BD</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Difoco LB Broth, Miller (Luria-Bertani)</td>
			<td>Becton Dickinson</td>
			<td style="width:119px;">244610</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DNA-free DNA Removal Kit&#160;</td>
			<td style="width:200px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">244510</td>
			<td>&#160;Dnase digestion</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Dried yeast</td>
			<td style="width:200px;">Rapunzel</td>
			<td style="width:119px;">&#160;-</td>
			<td>Organic cultivation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Dulbecco&#39;s Phosphate-Buffered Saline (DPBS)</td>
			<td>Thermo Fisher Scientific</td>
			<td style="width:119px;">14040</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Ethanol</td>
			<td style="width:200px;">VWR</td>
			<td style="width:119px;">20821.330</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Glycerol</td>
			<td>Sigma-Aldrich</td>
			<td style="width:119px;">W252506</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Honey</td>
			<td style="width:200px;">Osterm&#252;hle Naturkost GmbH</td>
			<td style="width:119px;">487</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Isopropanol&#160;</td>
			<td style="width:200px;">VWR</td>
			<td style="width:119px;">20842.330</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Lightcycler 480 Instrument II</td>
			<td style="width:200px;">Roche Molecular Systems</td>
			<td style="width:119px;">5015278001</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">LightCycler 480 Multiwell Plate 96, white</td>
			<td style="width:200px;">Roche Molecular Systems</td>
			<td style="width:119px;">4729692001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Manual Microsyringe Pump with Digital Display</td>
			<td>World Precision Instruments</td>
			<td style="width:119px;">DMP</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Micro-Fine+ U-100 insulin syringe 0.3 x 8 mm</td>
			<td>Becton Dickinson</td>
			<td style="width:119px;">324826</td>
			<td>Oral administration</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Mortar, unglazed</td>
			<td style="width:200px;">VWR</td>
			<td>410-9327&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Nanodrop</td>
			<td>Thermo Fisher Scientific</td>
			<td style="width:119px;">13-400-518</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Nuclease-free water&#160;</td>
			<td style="width:200px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">10977035</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Oxoid AnaeroGen sachets&#160;</td>
			<td>Thermo Fisher Scientific</td>
			<td style="width:119px;">AN0025A</td>
			<td>Quality and quantity of RNA</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">PCR stripes</td>
			<td style="width:200px;">Biozym</td>
			<td style="width:119px;">710970</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Pestle, unglazed grinding surface</td>
			<td style="width:200px;">VWR</td>
			<td>410-9324&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Phusion proof-reading enzyme&#160;</td>
			<td style="width:200px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">F553S</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Primers</td>
			<td style="width:200px;">Biomers</td>
			<td style="width:119px;">&#160;-</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">PureYield Plasmid Miniprep System</td>
			<td style="width:200px;">Promega</td>
			<td style="width:119px;">A1222</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">QuantiFast SYBR Green PCR kit&#160;</td>
			<td style="width:200px;">Qiagen</td>
			<td style="width:119px;">204056</td>
			<td>qPCR for bacterial copy number measurment</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">QuantiFast SYBR Green RT-PCR Kit&#160;</td>
			<td style="width:200px;">Qiagen</td>
			<td>204156</td>
			<td>qRT-PCR for gene expression measurements</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">QuantiTect Reverse Transcription Kit&#160;</td>
			<td style="width:200px;">Qiagen</td>
			<td style="width:119px;">205311</td>
			<td>cDNA synthesis</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Qubit Assay Tubes</td>
			<td style="width:200px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">Q32856</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Qubit dsHS DNA kit&#160;</td>
			<td style="width:200px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">Q32851</td>
			<td>Quantification of plasmid and cDNA samples</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Qubit fluorometer</td>
			<td style="width:200px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">Q33226</td>
			<td>Quantification of plasmid and cDNA samples</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:216px;">RNase-ExitusPlus</td>
			<td style="width:200px;">AppliChem</td>
			<td style="width:119px;">A7153</td>
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		<tr height="21">
			<td height="21" style="height:21px;">Rnasin Ribonuclease Inhibitor</td>
			<td style="width:200px;">Promega</td>
			<td style="width:119px;">N2511</td>
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		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Skimmed milk powder</td>
			<td>Sucofin</td>
			<td style="width:119px;">&#160;-</td>
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		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">SYBR safe DNA Gel Stain</td>
			<td style="width:200px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">S33102</td>
			<td></td>
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			<td height="21" style="height:21px;">TRI reagent&#160;</td>
			<td>Sigma-Aldrich</td>
			<td style="width:119px;">T9424</td>
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		</tr>
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			<td height="21" style="height:21px;">Weighing boat</td>
			<td>VWR</td>
			<td style="width:119px;">10803-148</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Wheat meal</td>
			<td style="width:200px;">Osterm&#252;hle Naturkost GmbH</td>
			<td style="width:119px;">6462</td>
			<td>Organic cultivation</td>
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a galleria mellonella oral administration model to study commensal-induced innate immune responses

The investigation of the immunogenic potential of commensal bacteria on the host immune system is one essential component when studying intestinal host-microbe interactions. It is well established that different commensals exhibit a different potential to stimulate the host intestinal immune system. Such investigations involve vertebrate animals, especially rodents. Since increasing ethical concerns are linked with experiments involving vertebrates, there is a high demand for invertebrate replacements models. 
Here, we provide a Galleria mellonella oral administration model using commensal non-pathogenic bacteria and the possible assessment of the immunogenic potential of commensals on the G. mellonella immune system. We demonstrate that G. mellonella is a useful alternative invertebrate replacement model that allows the analysis of commensals with different immunogenic potential such as Bacteroides vulgatus and Escherichia coli. Interestingly, the bacteria exhibited no killing effect on the larvae, which is similar to mammals. The immune responses of G. mellonella were comparable with vertebrate innate immune responses and involve recognition of the bacteria and production of antimicrobial molecules. We propose that G. mellonella was able to restore previous microbiota balance, which is well known from healthy mammalian individuals. Although providing comparable innate immune responses in both G. mellonella and vertebrates, G. mellonella does not harbor an adaptive immune system. Since the investigated components of the innate immune system are evolutionary conserved, the model allows a prescreening and first analysis of bacterial immunogenic properties.

The G. mellonella hemolymph infection model in widely used to analyze the virulence factors of a huge variety of pathogens. Most measurements include the analysis of larvae mortality, which is a quite easy method. Nevertheless, this method does not allow conclusions about immune responses in general and link the results of G. mellonella immune responses with vertebrate immune mechanisms. The G. mellonella oral administration model on the other hand is only rarel...

Watch this Scientific Journal Video about A Galleria mellonella Oral Administration Model to Study Commensal-Induced Innate Immune Responses at JoVE.com

A Galleria mellonella Oral Administration Model to Study Commensal-Induced Innate Immune Responses

Here, we provide a detailed protocol for an oral administration model using Galleria mellonella larvae and how to characterize induced innate immune responses. Using this protocol, researchers without practical experience will be able to use the G. mellonella force-feeding method.

A Galleria mellonella Oral Administration Model to Study Commensal-Induced Innate Immune Responses

The investigation of the immunogenic potential of commensal bacteria on the host immune system is one essential component when studying intestinal ...

Our protocol expands the use of G.mellonella from a systemic infection model to a model that allows bacterial administration via the natural oral route. G.mellonella is a suitable replacement model for rodents in analyzing different hallmarks of innate micro-post interactions as the larvae can be used for human pathogenic bacteria. Begin by transferring the eggs laid by adult moths to two liter boxes containing wax moth substrate, at 30 degrees Celsius, in the dark.After one to two weeks, transfer 25 grams of larva containing substrate into fresh substrate, when small and tiny larva become visible. Synchronize the larvae after two weeks according to their size, into groups of 30 to 40 larvae in new two liter containers on wax moth substrate. After two weeks, select larvae for the experiment by weight.Using only pale, fast moving larvae with a mass of 180 to 200 milligrams. To force feed the larvae, load an insulin syringe with a blunt-ended needle for oral application, with one times 10 to the seventh bacteria per 10 microliters of DPBS per larva. Load the syringe into a microsyringe pump.Wait for a larva to open its mouth parts before inserting the syringe carefully between the mandibles and delivering the bacteria. When all of the larva have been fed, incubate the animals in the dark at 37 degrees Celsius, between one to 24 hours. For larva processing, clean a safety hood with ethanol and spray reagent for preventing RNase contamination.Snap freeze the living larva in liquid nitrogen. Homogenize each individual frozen larva using a mortar and pestle and a small volume of liquid nitrogen. When a powdered homogenate has been produced, transfer the homogenate to a disposable wait boat and wait for the liquid nitrogen to evaporate.When all of the larva have been homogenized, mix the powdered homogenates with one milliliter of TRIzol in a two milliliter tube. Incubate the mixture at room temperature for one hour. At the end of the incubation, pellet the homogenate by centrifugation.Transfer the supernatant into a new tube. Mix the supernatant with 200 microliters of 1-Bromo-3-chloropropane, or BCP. Vortex and incubate for five minutes at room temperature, followed by a 10 minute incubation on ice.At the end of the second incubation, centrifuge the BCP added reaction and transfer the upper transparent layer into a new, two milliliter tube. Precipitate the RNA of the transferred upper layer with 500 microliters of isopropanol by mixing and inverting the tube for five minutes. Then collect the precipitate RNA by centrifugation.Wash the precipitated RNA pellet with 500 microliters of 75%ethanol and dry the RNA for five to 10 minutes at room temperature. When the ethanol has evaporated, add 100 microliters of ribonuclease inhibitor in nuclease-free water to the dried RNA pellet, and carefully vortex the tube until the pellet is completely dissolved. Measure the RNA quality and quantity by standard protocols, ensuring that the 260 to 280 ratio is approximately two, and the 260 to 230 ratio is in the range of two to 2.2.Then use five micrograms of the isolated RNA for DNase digestion and gene expression analysis, according to standard protocols. G.mellonella is able to clear the initial force fed bacterial load until no bacteria are detectable. With the 16s gene copy numbers of both B.vulgatus and E.coli substantially decreased within 24 hours.Commensal administered a G.mellonella larvae induce RNA gene expression of various innate immunity marker genes. For example, the LPS recognition molecules, apolipophorin and hemolin are generally expressed more highly in the E.coli administered larvae compared to B.vulgatus administered larvae. Further, the production of reactive oxygen and nitrogen species are strongly upregulated upon E.coli force feeding compared to B.vulgatus, as well as antioxidative GST gene expression.In addition, differential microbial peptide expression is induced more strongly after E.coli administration than in response to B.vulgatus force feeding. Remember to use only healthy, white, quickly moving larvae, to use patience during force feeding to avoid stressing the larvae and to include all of the necessary controls. The activation of G.mellonella immune markers can be determined directly by RUS or GST activity assays and the abundance of ANPs can be determined by bacterial growth inhibition assays.This technique can be used as a screening tool for commensal and pathogenic bacteria and for exploring intestinal symbiomes and pathobiomes without sacrifice of many vertebrates. Always use the appropriate personal protective equipment when working with liquid nitrogen, BCP, or TRIzol substances.

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Research

JoVE Journal

Immunology and Infection

7.8K visualizações.  University of Tübingen. Nosso protocolo expande o uso de G.mellonella de um modelo de infecção sistêmica para um modelo que permite a administração bacteriana através da rota oral natural. G.mellonella é um modelo de substituição adequado para roedores na análise de diferentes marcas de interações inatas de micro-pós, pois as larvas podem ser usadas para bactérias patogênicas humanas. Comece transferindo os ovos colocados por mariposas adultas para duas caixas de litro contendo substrato de mariposa d...