Name: the galleria mellonella waxworm infection model for disseminated candidiasis
Uploaded: 2018-11-17T15:00:00
Description: Watch this Scientific Journal Video about The Galleria mellonella Waxworm Infection Model for Disseminated Candidiasis at JoVE.com

The authors would like to acknowledge the assistance of Pamela Washington and Leah Anderson in obtaining Galleria mellonella for use in this study.

All methods described rely on use of invertebrate hosts and do not require Institutional Animal Care and Use Committee (IACUC) approval.
1. Galleria mellonella Waxworm Larvae
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	<li>Order larvae from wholesalers and suppliers that do not introduce hormones, antibiotics, or other treatments to the larvae and which are able to ship and deliver live specimens.
	<ol>
		<li>Be sure to purchase all larvae from the same supplier during the course of experimentation. Be careful when orde...

<ol>
	<li>Kauffman, C. 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=Prospective+multicenter+surveillance+study+of+funguria+in+hospitalized+patients.+The+National+Institute+for+Allergy+and+Infectious+Diseases+(NIAID)+Mycoses+Study+Group.">Prospective multicenter surveillance study of funguria in hospitalized patients. The National Institute for Allergy and Infectious Diseases (NIAID) Mycoses Study Group.</a> Clinical Infectious Diseases. 30 (1), 14-18 (2000).</li><li>Horn, D. 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=Epidemiology+and+outcomes+of+candidemia+in+2019+patients:+data+from+the+prospective+antifungal+therapy+alliance+registry.">Epidemiology and outcomes of candidemia in 2019 patients: data from the prospective antifungal therapy alliance registry.</a> Clinical Infectious Diseases. 48 (12), 1695-1703 (2009).</li><li>Pfaller, M. A., Diekema, 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=Epidemiology+of+invasive+candidiasis:+a+persistent+public+health+problem.">Epidemiology of invasive candidiasis: a persistent public health problem.</a> Clinical Microbiology Reviews. 20 (1), 133-163 (2007).</li><li>Sardi, J. C., Scorzoni, L., Bernardi, T., Fusco-Almeida, A. M., Mendes Giannini, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Candida+species:+current+epidemiology,+pathogenicity,+biofilm+formation,+natural+antifungal+products+and+new+therapeutic+options.">Candida species: current epidemiology, pathogenicity, biofilm formation, natural antifungal products and new therapeutic options.</a> Journal of Medical Microbiology. 62, 10-24 (2013).</li><li>Segal, E., Frenkel, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Experimental+in+Vivo+Models+of+Candidiasis.">Experimental in Vivo Models of Candidiasis.</a> J Fungi (Basel). 4 (1), (2018).</li><li>Conti, H. R., Huppler, A. R., Whibley, N., Gaffen, S. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Animal+models+for+candidiasis.">Animal models for candidiasis.</a> Current Protocols in Immunology. 105, 11-17 (2014).</li><li>Heymann, P., 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+siderophore+iron+transporter+of+Candida+albicans+(Sit1p/Arn1p)+mediates+uptake+of+ferrichrome-type+siderophores+and+is+required+for+epithelial+invasion.">The siderophore iron transporter of Candida albicans (Sit1p/Arn1p) mediates uptake of ferrichrome-type siderophores and is required for epithelial invasion.</a> Infection and Immunity. 70 (9), 5246-5255 (2002).</li><li>Priest, S. J., Lorenz, M. 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W., Frankel, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+Galleria+mellonella+as+a+model+organism+to+study+Legionella+pneumophila+infection.">Use of Galleria mellonella as a model organism to study Legionella pneumophila infection.</a> Journal of Visualized Experiments. (81), e50964 (2013).</li><li>Jacobsen, I. D. <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+host+to+study+virulence+of+Candida.">Galleria mellonella as a model host to study virulence of Candida.</a> Virulence. 5 (2), 237-239 (2014).</li><li>Tsai, C. J., Loh, J. 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Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+diversification+accompanies+gene+family+expansion+of+MED2+homologs+in+Candida+albicans.">Functional diversification accompanies gene family expansion of MED2 homologs in Candida albicans.</a> PLoS Genetics. 14 (4), (2018).</li><li>Astvad, K. M. T., Meletiadis, J., Whalley, S., Arendrup, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluconazole+Pharmacokinetics+in+Galleria+mellonella+Larvae+and+Performance+Evaluation+of+a+Bioassay+Compared+to+Liquid+Chromatography-Tandem+Mass+Spectrometry+for+Hemolymph+Specimens.">Fluconazole Pharmacokinetics in Galleria mellonella Larvae and Performance Evaluation of a Bioassay Compared to Liquid Chromatography-Tandem Mass Spectrometry for Hemolymph Specimens.</a> Antimicrobial Agents and Chemotherapy. 61 (10), (2017).</li><li>Mesa-Arango, A. 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=The+non-mammalian+host+Galleria+mellonella+can+be+used+to+study+the+virulence+of+the+fungal+pathogen+Candida+tropicalis+and+the+efficacy+of+antifungal+drugs+during+infection+by+this+pathogenic+yeast.">The non-mammalian host Galleria mellonella can be used to study the virulence of the fungal pathogen Candida tropicalis and the efficacy of antifungal drugs during infection by this pathogenic yeast.</a> Med Mycol. 51 (5), 461-472 (2013).</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 Immunology and Medical Microbiology. 34 (2), 153-157 (2002).</li><li>Fuchs, B. B., O'Brien, E., Khoury, J. B., Mylonakis, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Methods+for+using+Galleria+mellonella+as+a+model+host+to+study+fungal+pathogenesis.">Methods for using Galleria mellonella as a model host to study fungal pathogenesis.</a> Virulence. 1 (6), 475-482 (2010).</li><li>Kavanagh, K., Fallon, J. P. <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+larvae+as+models+for+studying+fungal+virulence.">Galleria mellonella larvae as models for studying fungal virulence.</a> Fungal Biology Reviews. 24 (1-2), 79-83 (2010).</li><li>Hull, C. M., Johnson, A. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+a+mating+type-like+locus+in+the+asexual+pathogenic+yeast+Candida+albicans.">Identification of a mating type-like locus in the asexual pathogenic yeast Candida albicans.</a> Science. 285 (5431), 1271-1275 (1999).</li><li>Legrand, M., 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=Homozygosity+at+the+MTL+locus+in+clinical+strains+of+Candida+albicans:+karyotypic+rearrangements+and+tetraploid+formation.">Homozygosity at the MTL locus in clinical strains of Candida albicans: karyotypic rearrangements and tetraploid formation.</a> Molecular Microbiology. 52 (5), 1451-1462 (2004).</li><li>Lockhart, S. 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=In+Candida+albicans,+white-opaque+switchers+are+homozygous+for+mating+type.">In Candida albicans, white-opaque switchers are homozygous for mating type.</a> Genetics. 162 (2), 737-745 (2002).</li><li>Miller, M. G., Johnson, A. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=White-opaque+switching+in+Candida+albicans+is+controlled+by+mating-type+locus+homeodomain+proteins+and+allows+efficient+mating.">White-opaque switching in Candida albicans is controlled by mating-type locus homeodomain proteins and allows efficient mating.</a> Cell. 110 (3), 293-302 (2002).</li><li>Wu, W., Lockhart, S. R., Pujol, C., Srikantha, T., Soll, D. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heterozygosity+of+genes+on+the+sex+chromosome+regulates+Candida+albicans+virulence.">Heterozygosity of genes on the sex chromosome regulates Candida albicans virulence.</a> Molecular Microbiology. 64 (6), 1587-1604 (2007).</li><li>Herrero, A. B., 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=KRE5+gene+null+mutant+strains+of+Candida+albicans+are+avirulent+and+have+altered+cell+wall+composition+and+hypha+formation+properties.">KRE5 gene null mutant strains of Candida albicans are avirulent and have altered cell wall composition and hypha formation properties.</a> Eukaryotic Cell. 3 (6), 1423-1432 (2004).</li><li>Hall, R. 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=The+Mnn2+mannosyltransferase+family+modulates+mannoprotein+fibril+length,+immune+recognition+and+virulence+of+Candida+albicans.">The Mnn2 mannosyltransferase family modulates mannoprotein fibril length, immune recognition and virulence of Candida albicans.</a> PLoS Pathogens. 9 (4), 1003276 (2013).</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> Journal of Insect Science. 24 (3), 342-357 (2017).</li><li>Bergin, D., Reeves, E. P., Renwick, J., Wientjes, F. B., 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=Superoxide+production+in+Galleria+mellonella+hemocytes:+identification+of+proteins+homologous+to+the+NADPH+oxidase+complex+of+human+neutrophils.">Superoxide production in Galleria mellonella hemocytes: identification of proteins homologous to the NADPH oxidase complex of human neutrophils.</a> Infection and Immunity. 73 (7), 4161-4170 (2005).</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=Genome+Sequence+of+Galleria+mellonella+(Greater+Wax+Moth).">Genome Sequence of Galleria mellonella (Greater Wax Moth).</a> Genome Announcements. 6 (2), (2018).</li><li>Krappmann, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lightning+up+the+worm:+How+to+probe+fungal+virulence+in+an+alternative+mini-host+by+bioluminescence.">Lightning up the worm: How to probe fungal virulence in an alternative mini-host by bioluminescence.</a> Virulence. 6 (8), 727-729 (2015).</li><li>Chowdhary, A., Voss, A., Meis, J. 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The G. mellonella waxworm model stands as an effective tool for the rapid and reproducible analysis of C. albicans virulence. This detailed protocol relies upon consistent delivery of a defined infectious dose to the same site across a batch of larvae. Infectious dose has a profound impact on G. mellonella mortality whereas use of larvae between their initial arrival and ten days following receipt produced similar results. Loss of the C. albicansMTLa allele results in ...

Candida species are common human commensals that are capable of emerging as opportunistic pathogens in severely immunocompromised and dysbiotic patients. Although many Candida species can cause disease, C. albicans is the most prevalent cause of disseminated candidiasis1,2. Systemic disease results from C. albicans accessing the bloodstream through either direct penetration of previously restrictive host barriers or introduction at surgical sites and other breaches of the body3. Candida species utilize a range of pathogenic processes to cause systemic disease within the host including filamentation, biofilm formation, immune cell evasion and escape, and iron scavenging4. In vitro approaches exist to investigate individual pathogenic mechanisms, but animal models continue to provide the best option to investigate the entirety of disease outcome5,6. Previous research has detailed many instances of promising in vitro investigations of virulence failing to reproduce in vivo7,8. Thus, animal models are still required to assess virulence in vivo. Most disease models rely on mice to serve as a surrogate for human infections despite C. albicans inability to naturally colonize murine systems as a commensal9. Invertebrate models of disseminated candidiasis include the nematode Caenorhabditis elegans, the fruit fly Drosophila melanogaster, and the waxworm Galleria mellonella, although concerns about fundamental differences in basic physiology, host body temperatures, and routes of exposure have hampered their broad acceptance10,11. 
Most recently, the G. mellonella waxworm infection model has been adopted to model pathogenicity of a wide range of bacterial and fungal pathogens12,13,14. Advantages of this model include its relatively low cost, increased throughput, ease of use, and reduced ethical concerns regarding animal beneficence compared to murine models. For researchers, this translates into increased ability to test multiple variables, stronger confidence intervals, more rapid experimentation, and bypass of animal protocols. G. mellonella has served as a platform to rapidly assess C. albicans virulence following perturbation of genes required for biofilm formation, filamentation, and gene regulation across clinical isolates11,15,16. Recent studies have incorporated investigation of antifungal efficacy using G. mellonella to assess the pharmacokinetics of drug activity and resistance under in vivo settings, which are otherwise challenging and time consuming17,18. Yet, studies of C. albicans virulence in G. mellonella have been complicated by reportedly high levels of variation within experiments and inconsistent protocols between research groups that produce differing virulence phenotypes between mice and waxworms11,13,19,20,21. Here, we outline a G. mellonella protocol to standardize C. albicans infections, increase reproducibility in virulence experiments, and demonstrate consistency with previously described studies of virulence in murine models.
Previous studies demonstrated that the C. albicans mating type-like (MTL) locus on chromosome 5 regulates cell identity and mating competence similar to Saccharomyces cerevisiae and other Ascomycete fungi22. The majority of C. albicans isolates are heterozygous at the MTL locus, encoding one of each of the MTLa&#160;and MTL&#945; alleles (MTLa/&#945;), and are consequently sterile15,23,24. Loss of one of the MTL alleles through loss of heterozygosity (LOH) or mutation leads to homozygous MTLa or MTL&#945; strains that can undergo a phenotypic switch from the sterile &#39;white&#39; state to the mating competent &#39;opaque&#39; state25. Previous work has highlighted that loss of MTL heterozygosity also reduces virulence in murine models of systemic infection across different strain backgrounds26. Here, we detail the G. mellonella model for disseminated candidiasis using a genetically similar experimental set to depict the contribution of MTL heterozygosity to virulence in G. mellonella. We show that MTL configuration influenced C. albicans pathogenicity, where MTL&#945; strains were less virulent with respect to both MTLa/&#945; and MTLa cells, similar to findings within murine infection model26.

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	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:427px;">Galleria mellonella</td>
			<td style="width:140px;">Snackworms.com</td>
			<td style="width:119px;"></td>
			<td style="width:167px;">Buy twice as many worms as expected to use</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">10 uL, Model 1701 N SYR Cemented needle, 26G, type 2 syringe</td>
			<td>Hamilton</td>
			<td>80000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Petri dish, 100X15 mm, 500 pack</td>
			<td>Fisher</td>
			<td>FB0875712</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Microcentrifuge tube, 1.7 mL, 500 pack</td>
			<td style="width:140px;">VWR</td>
			<td>87003-294</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:427px;">Phosphate Buffered Saline (Biotechnology grade), 500 mL</td>
			<td style="width:140px;">VWR</td>
			<td style="width:119px;">97062-818</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:427px;">Ethanol absolute, &#8805;99.5% pure, 500 mL</td>
			<td style="width:140px;">Millipore Sigma</td>
			<td>EM-EX0276-1S</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:427px;">autoclaved ddH2O</td>
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the galleria mellonella waxworm infection model for disseminated candidiasis

Candida species are common fungal commensals of humans colonizing the skin, mucosal surfaces, and gastrointestinal tract. Under certain conditions, Candida can overgrow their natural niches resulting in debilitating mucosal infections as well as life-threatening systemic infections, which are a major focus of investigation due to their associated high mortality rates. Animal models of disseminated infection exist for studying disease progression and dissecting the characteristics of Candida pathogenicity. Of these, the Galleria mellonella waxworm infection model provides a cost-effective experimental tool for high-throughput investigations of systemic virulence. Many other bacterial and eukaryotic infectious agents have been effectively studied in G. mellonella to understand pathogenicity, making it a widely accepted model system. Yet, variation in the method used to infect G. mellonella can alter phenotypic outcomes and complicate interpretation of the results. Here, we outline the benefits and drawbacks of the waxworm model to study systemic Candida pathogenesis and detail an approach to improve reproducibility. Our results highlight the range of mortality kinetics in G. mellonella and describe the variables which can modulate these kinetics. Ultimately, this method stands as an ethical, rapid, and cost-effective approach to study virulence in a model of disseminated candidiasis.

Here, we demonstrate a reproducible method for the use of G. mellonella waxworms to investigate a disseminated candidiasis model of infection using C. albicans. The appropriate storage, maintenance, and selection of larvae for infection are critical component of insuring reproducibility in G. mellonella mortality (Figure 1A). Healthy larvae that are active, have a light yellow/tan color, and lack black patches on th...

Watch this Scientific Journal Video about The Galleria mellonella Waxworm Infection Model for Disseminated Candidiasis at JoVE.com

The Galleria mellonella Waxworm Infection Model for Disseminated Candidiasis

Galleria mellonella serves as an invertebrate model for disseminated candidiasis. Here, we detail the infection protocol and provide supporting data for the model&#39;s effectiveness.

The Galleria mellonella Waxworm Infection Model for Disseminated Candidiasis

Candida species are common fungal commensals of humans colonizing the skin, mucosal surfaces, and gastrointestinal tract. Under certain conditions, ...

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Use of the Invertebrate Galleria mellonella as an Infection Model to Study the Mycobacterium tuberculosis Complex

Use of the Invertebrate Galleria mellonella as an Infection Model to Study the Mycobacterium tuberculosis Complex

Tuberculosis is the leading global cause of infectious disease mortality and roughly a quarter of the world&#8217;s population is believed to be infected ...

Tools for the Real-Time Assessment of a Pseudomonas aeruginosa Infection Model

Tools for the Real-Time Assessment of a Pseudomonas aeruginosa Infection Model

Pseudomonas aeruginosa (Pa) is one of the most common opportunistic pathogens associated with cystic fibrosis (CF). Once Pa colonization is established, a ...

A Mouse Ear Model for Allergic Contact Dermatitis Evaluation

Skin is the human body&#39;s first line of defense and one of the most exposed organs to environmental chemicals. Allergic contact dermatitis (ACD) is a ...