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
<ol>
	<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 ordering larvae during summer months as temperatures exceeding 30 &#176;C decrease larvae viability.</li>
		<li>Monitor temperatures across the expected shipping route and plan shipping and delivery accordingly or choose expedited shipping to minimize their exposure to environmental conditions.</li>
	</ol>
	</li>
	<li>When larvae arrive, check each container to ensure viable, healthy larvae are present. Healthy larvae will be completely submerged beneath the bedding, moving, and possess a light yellow/tan coloration with few larvae containing black marks or discolorations along the body.</li>
	<li>Store larvae in their container in a ventilated space at ambient temperature (25 &#176;C). Depending on the initial condition of the larvae, they may be used for up to two weeks.</li>
</ol>
2. Culture Preparation for Galleria mellonella Infection
NOTE: Proper lab attire should be worn throughout this portion of the protocol including gloves, lab coat, and safety glasses.
<ol>
	<li>Make yeast peptone dextrose (YPD) liquid and agar plates. Prepare 1x phosphate buffered saline (PBS) and 70% ethanol solutions.</li>
	<li>Grow C. albicans strains to be injected overnight in 3 mL of fresh YPD in standard culture tubes on a rotating drum at medium speed and 30 &#176;C.</li>
	<li>Spin cells down at 2,500 x g for 5 min in a tabletop centrifuge. Pour off supernatant, being careful not to disturb the cell pellet.
	<ol>
		<li>Resuspend cells fully in 5 mL of 1x PBS by repeated pipetting or vortexing. Repeat the centrifugation and resuspension of cells in PBS twice more to ensure removal of all culture media.</li>
	</ol>
	</li>
	<li>After a final wash, resuspend cells in 1 mL of PBS and transfer them to a microcentrifuge tube.</li>
	<li>Conduct a set of serial dilutions in PBS to generate a 1:100, 1:1,000, and 1:100,000 dilution series. 
	&#8203;NOTE: Alternative dilutions may be necessary to reach desired cell counts.
	<ol>
		<li>Using a hemocytometer, count cells from either the 1:100 or the 1:1,000 dilution. Most often, the 1:1,000 dilution provides the appropriate cell counts for C. albicans cultures.</li>
		<li>Calculate total concentration of cells within the initial culture using the grid math of the hemocytometer, aiming for between 30-300 cells to be counted in the central 5x5 grid. An automated cell counter may be used as an alternative; however, use of optical density to determine cell counts is discouraged as cell morphology (size, shape, etc.) can significantly affect its accuracy.</li>
	</ol>
	</li>
	<li>Using the measured cell counts, make a new dilution of 2.5x107 cells/mL in 1x PBS in a microcentrifuge tube. This dilution will serve as the basis for each inoculation of G. mellonella with C. albicans. Each injection will require 10 &#181;L of this inoculum for an infectious dose of 250,000 C. albicans cells.</li>
	<li>Confirm accuracy of the infectious dose by plating the correct volume of the 1:100,000 dilution for 100 colony forming units (CFUs) on YPD agar for each culture to be used in infection.</li>
	<li>Incubate cell count plates from step 2.7 for 48 hours (h) at 30 &#176;C and count the number of colonies per plate. Between 80 and 120 colonies constitutes an acceptable range for accuracy in the inoculum.</li>
</ol>
3. Infection of Galleria mellonella Larvae with Candida Cultures
NOTE: Proper lab attire should be worn throughout this portion of the protocol including gloves, lab coat, and safety glasses.
<ol>
	<li>Add 1 mL of 100% ethanol and 1 mL of 1x PBS to two separate microcentrifuge tubes. The ethanol and PBS will be used to wash the injection needle between infections.</li>
	<li>Spread a small amount of waxworm bedding in an empty, 100 x 15 mm sterile Petri dish, which will house the larvae following inoculation for each independent strain. Addition of the bedding limits adherence of dead G. mellonella larvae to the Petri dish surface.</li>
	<li>Sterilize a 26 G, 10 &#181;L syringe by taking up and expunging 10 &#181;L of 70% ethanol three times followed by three washes with 10 &#181;L of 1x PBS. Vortex the inoculation dilution of C. albicans and take up 10 &#181;L of culture. Ensure that there are no air bubbles within the syringe as injection of air promotes death and will complicate downstream analysis.</li>
	<li>Open a container holding the G. mellonella larvae and carefully turn bedding over with a finger to uncover larvae. Select larvae which are in good health as identified by active movement, a light yellow/tan color, and a lack of black pigmentation on the larvae body. These larvae should be of similar size across the full experimental set.</li>
	<li>Pick up a single larvae and hold it gently between the index/middle finger and thumb similar to holding a pencil. Roll the larvae onto its back so the legs are facing up. Hold the full length of the body between the fingers and thumb so that the larvae is unable to curl or pull away from the injection. 
	&#8203;NOTE: If desired, sterilize the site of injection using a cotton swab soaked in 100% ethanol.</li>
	<li>Rotate the syringe so the needle&#39;s bevel faces up and slowly insert the needle tip including the length of the bevel into the body at the junction with the rearmost left leg. Ensure that the needle penetrates the body and does not simply push the body of the larvae inwards. Do not use force in penetrating the body as the needle may pierce completely through the larvae quickly. Gently lifting with the needle will confirm appropriate penetration. Inject the full 10 &#181;L Candida inoculum and extract the needle.</li>
	<li>Repeat step 3.3-3.5 for each larvae. To prevent cell settling, vortex the Candida cultures for inoculation after every third injection. As a control, inject a set of G. mellonella with 10 &#181;L of 1X PBS.</li>
	<li>Co-house 10 larvae injected from the same Candida culture in each 100 x 15 mm Petri dish following inoculation. Incubate larvae at 37 &#176;C for eight days, checking larvae every 24 h to monitor death.
	<ol>
		<li>Assess mortality initially by darkened pigmentation, the formation of black patches or bodies, and a lack of movement. To confirm mortality, use tweezers to gently roll moribund larvae onto their back and lightly poke the larvae&#39;s underside with the tweezers. Non-responsiveness as observed by a lack of visible body or leg movement is scored as death and the larvae are removed from the Petri dish.</li>
	</ol>
	</li>
	<li>Record larvae mortality over the time course. Larvae that begin to pupate into moths can be included in the analysis but should be removed if they begin to molt. Pupating larvae can be censored from end point analysis as the virulence differences between larvae and pupa are unclear.</li>
	<li>Assess statistical significance using Mantel-Cox statistical test.</li>
</ol>

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The authors have nothing to disclose.

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 decreased virulence consistent with previous experiments in mice although disruption of the MTL&#945; allele did not alter larvae death.
The outcome of G. mellonella infections can be significantly impacted by the experimental design. In contrast to vertebrate models of infection, small variation in the injection site, inoculum volume, and inoculum size may impact interpretations of the data. Infections with increasing numbers of C. albicans cells led to greater and more rapid morbidity of infected larvae. At a certain point, injecting too many C. albicans appears capable of overwhelming host defenses leading to rapid death of the host, potentially through toxic shock. However, SC5314 was consistently the most virulent strain at all doses, followed by 12C, and then the other strains. Thus, infection parameters have a profound impact on the absolute kinetics of infections but may still provide estimates of relative virulence. Prior work within these experiments further demonstrated the importance of thoroughly washing the Candida cells. Residual YPD included in the injection bolus significantly decreases larvae survival (data not shown).
Worm health is a critical variable when conducting G. mellonella experiments. Larvae transported using standard shipping in the middle of summer often arrive prematurely aged or stressed, covered with black patches, with decreased viability. No significant role for larvae size on morbidity has been previously observed although the experiments described here focused on assaying larvae of roughly equivalent mass. Healthy larvae and PBS control larvae should always be run alongside infection groups to account for any changes in larvae viability between biological replicates performed on separate days. G. mellonella infected ten days after arriving matched the kinetics of larvae infected immediately upon receipt. Thus, a single shipment of larvae can provide sufficient material and time to perform full experiments. These experiments relied upon 30 animals per strain to determine differences in virulence, which is significantly more animals than typically utilized in murine models, resulting in stronger confidence of observed results11,27,28. This model also reduced ethical concerns, time, and costs compared to vertebrate models of infection. Thus, the use of wax larvae may facilitate identification of smaller effects on systemic virulence than is possible in other systems.
Inherent caveats to the G. mellonella system exist that limit its utility in studying host-pathogen interactions. First, G. mellonella lack an adaptive immune system and cannot be used to investigate antigenicity or immunological memory as in higher eukaryotes29. The body of the larvae is composed of a single continuous cavity filled with hemolymph, which function to circulate nutrients, signaling molecules, immune cells, and antimicrobial peptides. These immune cells, hemocytes, behave similarly to neutrophils and can therefore phagocytose, generate oxidative bursts, and secrete lytic enzymes following activation through extracellular pathogen recognition receptors30. Yet, this function is a subset of innate immune cell processes within chordate systems that may not fully reflect all leukocyte activities. Another major caveat is the lack of a genome assembly for G. mellonella although the genome was recently sequenced31. Consequently, the extent of genotypic diversity within the species is unknown and most suppliers likely ship genetically heterogeneous populations that may affect mortality across experiments. Furthermore, molecular techniques do not exist for the organism and complicate any attempts to dissect host contributions to pathogenesis. Yet, consistent virulence results following infection with C. albicans across experiments, vendors, time, and labs supports their utility in fundamental questions of pathogenicity.
Loss of MTLa reduced virulence of C. albicans relative to MTL heterozygotes and MTL&#945; homozygote backgrounds. This trend is consistent with prior work performed using mouse models of systemic virulence26. In contrast to murine models, loss of MTL&#945; did not alter virulence relative to the MTLa/&#945; parental strain. The discrepancy between these results is unclear but may include components of adaptive immunity that are missing in invertebrate organisms. Alternatively, increased virulence associated with MTL heterozygosity may be attributable only to the MTL&#945; allele in SC5314, potentiating the need for assessment across multiple strain backgrounds. Therefore, we suggest a differential role for virulence between the two MTL alleles.
Overall, the G. mellonella model serves as a rapid and reproducible model for assessing systemic candidiasis. Other readouts of virulence can be assayed with this model including fungal prevalence by plating larvae homogenate for colony forming units (CFUs)11 or bioluminescence, which also provides fungal localization32. In the future, this model can be expanded to assess virulence characteristics of other Candida species such as elevated drug resistance in the newly emergent Candida auris species33. Continued development of tools to visualize lymphocyte within G. mellonella interacting with infecting Candida, inbred waxworm lineages to reduce variability in infections outcomes, and visualization of the infective process via reporter Candida lines34 will further refine this model of disease and allow deeper investigations of in vivo host-pathogen interactions.

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>
			<td style="width:140px;"></td>
			<td style="width:119px;"></td>
			<td></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 the body should be used for this system and are typically viable for infection up to two weeks following arrival. To minimize any potentially confounding effects of G. mellonella physiology, larvae were selected of similar size and weight. A well-mixed inoculum and consistent injection at the same anatomical site of the larvae body also stand as important variables in promoting reproducibility between experiments. Images showcase maintenance of infected larvae through the duration of experimentation (Figure 1B).
To assess the impact of larvae age on virulence, separate PBS control infections were performed with larvae either zero or ten days after arriving. No significant difference in the kinetics of G. mellonella death existed between these groups (Figure 2A). Characteristic signs of illness arose in larvae from both groups that succumbed to the infection; the originally yellow/tan coloration of the larvae became discolored and gave way to black patches before total loss of motility and, eventually, death (Figure 2B). Infection with C. albicans speeds up this process of discoloration and morbidity but does not significantly alter the cascade of phenotypic markers leading to death compared to uninfected larvae or PBS injected larvae (Figure 2C).
The infective dose also significantly impacts mortality kinetics of G. mellonella infections. To assess the effects of C. albicans inoculum size, we infected G. mellonella with four different clinical isolates (12C, P60002, P75010, and SC5314: Table 1) at three different doses. Larvae infected with SC5314 displayed mortality at all doses, consistent with increased pathogenicity of this isolate relative to most others used within the experiment (Figure 2D). More larvae succumbed to infection with 1.0 x 105 SC5314 or 12C cells compared to 1.0 x 104 cells. The highest dose of 1.0 x 106 cells proved excessively lethal with larvae dying following infection of all strains including the less virulent isolates, P60002 and 12C. Larvae injected with P75010 and SC5314 succumbed to the infection within 24 h, suggesting a more moderate infectious dose is appropriate to determine differences in virulence. Consequently, an infectious dose of 2.5x105 was used for all subsequent experiments.
To demonstrate similarity in virulence between murine and G. mellonella models of systemic disease, we assayed virulence of SC5314-derived C. albicans strains encoding either heterozygous (a/&#945; or homozygous (a/-or &#945;/-) MTL loci. Infection of G. mellonella with MTL heterozygotes from either the SC5314 or its prototrophic derivative BWP17 genetic background produced high rates of mortality compared to PBS-injected animals. Three-quarters of the control larvae survived to day 8 compared to loss of over half of all C. albicans injected animals within 3 days post infection (dpi) that further increased at 8 dpi (Figure 3A, 3B). Infection with BWP17 dampened C. albicans lethality in this model (27% survival compared to 4% survival in SC5314 MTL heterozygotes at 8 dpi; Figure 3C, 3D). G. mellonella larvae infected with MTL&#945;/- strains survived significantly longer compared to the MTL heterozygous parental strains (log-rank test, SC5314; p=0.0006, BWP17; p=0.0002). Interestingly, virulence of MTLa/- C. albicans cells matched its MTL heterozygous counterpart in both the SC5314 and BWP17 backgrounds. Thus, MTL configuration does have an influence on C. albicans virulence with MTL&#945;/- strains displaying decreased killing compared to either MTLa/&#945; or MTLa/- cells.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/58914/58914fig1.jpg" /> 
Figure 1. Overview of infection procedure. (A) A flowchart highlights the key elements of the infection procedure including ordering and storage of larvae, infection culture preparation, and injection. (B) Representative images show healthy storage conditions, preparation of the infection space, injection, and larvae maintenance following infection. <a href="https://www.jove.com/files/ftp_upload/58914/58914fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/58914/58914fig2.jpg" /> 
Figure 2. The effects of G. mellonella infection procedures. (A) PBS controls were infected either immediately upon arrival (PBS-D0, blue) or after 10 days (PBS-D10, red). (B) Representative images highlight larvae discoloration and death. (C) An image series of infection plates detail the effects of injection with PBS (middle) or with SC5314 (lower) compared to uninfected larvae (top) on days 1, 3, 5, and 7 following injection. (D) Dosage effects are shown within a collection of clinical isolates. <a href="https://www.jove.com/files/ftp_upload/58914/58914fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/58914/58914fig3.jpg" /> 
Figure 3. The MTLa allele promotes C. albicans killing of G. mellonella. MTL locus heterozygous (a/&#945;) and homozygous (a/- and &#945;/-) SC5314 (A) and BWP17 (C) derivatives are plotted for larvae mortality over eight days. Confidence intervals are plotted for each SC5314 (B) and BWP17 (D) derivative strain to highlight consistency in the kinetics of larvae mortality across experiments. The average (solid line) is plotted with standard deviations (filled in space) for three biological replicates of 10 larvae each. <a href="https://www.jove.com/files/ftp_upload/58914/58914fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table fo:keep-together.within-page="1">
	<tbody>
		<tr>
			<td>Candida strain</td>
			<td>Parental strain</td>
			<td>Genotype</td>
			<td>MTL</td>
			<td>Reference</td>
		</tr>
		<tr>
			<td>MAY1</td>
			<td>SC5314</td>
			<td>Candida albicans clinical isolate</td>
			<td>a/&#945;</td>
			<td>Wu et al. 2007</td>
		</tr>
		<tr>
			<td>MAY6</td>
			<td>P60002</td>
			<td>Candida albicans clinical isolate</td>
			<td>a/a</td>
			<td>Wu et al. 2007</td>
		</tr>
		<tr>
			<td>MAY11</td>
			<td>P75010</td>
			<td>Candida albicans clinical isolate</td>
			<td>a/&#945;</td>
			<td>Wu et al. 2007</td>
		</tr>
		<tr>
			<td>MAY15</td>
			<td>12C</td>
			<td>Candida albicans clinical isolate</td>
			<td>a/a</td>
			<td>Wu et al. 2007</td>
		</tr>
		<tr>
			<td>MAY61</td>
			<td>SC5314</td>
			<td>MTLa::SAT1</td>
			<td>&#945;/-</td>
			<td>This study</td>
		</tr>
		<tr>
			<td>MAY71</td>
			<td>SC5314</td>
			<td>MTL&#945;::SAT1S tac1::TAC1-GFP-HYGR</td>
			<td>a/-</td>
			<td>This study</td>
		</tr>
		<tr>
			<td>MAY313</td>
			<td>BWP17</td>
			<td>ura3&#916;::&#955;imm434/ura3&#916;::&#955;imm434 his1::hisG/his1::hisG arg4::hisG/arg4::RS-arg4-ura3-BN sir2::his1/sir2::dpl200</td>
			<td>a/&#945;</td>
			<td>This study</td>
		</tr>
		<tr>
			<td>MAY314</td>
			<td>BWP17</td>
			<td>ura3&#916;::&#955;imm434/ura3&#916;::&#955;imm434 his1::hisG/his1::hisG arg4::hisG/arg4::RS-ARG4-URA3-BN sir2::HIS1/sir2::dpl200</td>
			<td>&#945;/-</td>
			<td>This study</td>
		</tr>
		<tr>
			<td>MAY315</td>
			<td>BWP17</td>
			<td>ura3&#916;::&#955;imm434/ura3&#916;::&#955;imm434 his1::hisG/his1::hisG arg4::hisG/arg4::RS-ARG4-URA3-BN sir2::HIS1/sir2::dpl200</td>
			<td>a/-</td>
			<td>This study</td>
		</tr>
	</tbody>
</table>
Table 1. C. albicans strains used in this study.
<table fo:keep-together.within-page="1">
	<tbody>
		<tr>
			<td>Variable component</td>
			<td>Suggested control</td>
			<td>Reference</td>
		</tr>
		<tr>
			<td>Larvae age at time of injection</td>
			<td>Minimize time between worm arrival and inoculation</td>
			<td>This study</td>
		</tr>
		<tr>
			<td>Larvae size at time of injection</td>
			<td>Ensure consistency when selecting worms</td>
			<td>Fuchs et al. 2010</td>
		</tr>
		<tr>
			<td>Larvae health at time of injection</td>
			<td>Order worms during moderate temperature, use immediately upon arrival</td>
			<td>This study</td>
		</tr>
		<tr>
			<td>Infectious dose with C. albicans</td>
			<td>Select an infectious concentration which presents widest range of outcomes</td>
			<td>This study</td>
		</tr>
		<tr>
			<td>Larvae response to injection</td>
			<td>Conduct PBS injection controls alongside worms with no injection</td>
			<td>Hirakawa et al. 2015</td>
		</tr>
	</tbody>
</table>
Table 2. Considerations regarding G. mellonella infection.

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, ...

the-galleria-mellonella-waxworm-infection-model-for-disseminated

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JoVE Journal

Immunology and Infection

11.0K Views.  The Ohio State University. Galleria mellonella serves as an invertebrate model for disseminated candidiasis. Here, we detail the infection protocol and provide supporting data for the model's effectiveness.

Video: The Galleria mellonella Waxworm Infection Model for Disseminated Candidiasis

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 ...

Non-invasive Imaging of Disseminated Candidiasis in Zebrafish Larvae

Disseminated candidiasis caused by the pathogen Candida albicans is a clinically important problem in hospitalized individuals and is associated with a 30 to 40% attributable mortality6. Systemic candidiasis is normally controlled by innate immunity, and individuals with genetic defects in innate immune cell components such as phagocyte NADPH oxidase are more susceptible to candidemia7-9. Very little is known about the dynamics of C. albicans interaction with innate immune cells in vivo. Extensive in vitro studies have established that outside of the host C. albicans germinates inside of macrophages, and is quickly destroyed by neutrophils10-14. In vitro studies, though useful, cannot recapitulate the complex in vivo environment, which includes time-dependent dynamics of cytokine levels, extracellular matrix attachments, and intercellular contacts10, 15-18. To probe the contribution of these factors in host-pathogen interaction, it is critical to find a model organism to visualize these aspects of infection non-invasively in a live intact host. 
The zebrafish larva offers a unique and versatile vertebrate host for the study of infection. For the first 30 days of development zebrafish larvae have only innate immune defenses2, 19-21, simplifying the study of diseases such as disseminated candidiasis that are highly dependent on innate immunity. The small size and transparency of zebrafish larvae enable imaging of infection dynamics at the cellular level for both host and pathogen. Transgenic larvae with fluorescing innate immune cells can be used to identify specific cells types involved in infection22-24. Modified anti-sense oligonucleotides (Morpholinos) can be used to knock down various immune components such as phagocyte NADPH oxidase and study the changes in response to fungal infection5. In addition to the ethical and practical advantages of using a small lower vertebrate, the zebrafish larvae offers the unique possibility to image the pitched battle between pathogen and host both intravitally and in color. 
The zebrafish has been used to model infection for a number of human pathogenic bacteria, and has been instrumental in major advances in our understanding of mycobacterial infection3, 25. However, only recently have much larger pathogens such as fungi been used to infect larva5, 23, 26, and to date there has not been a detailed visual description of the infection methodology. Here we present our techniques for hindbrain ventricle microinjection of prim25 zebrafish, including our modifications to previous protocols. Our findings using the larval zebrafish model for fungal infection diverge from in vitro studies and reinforce the need to examine the host-pathogen interaction in the complex environment of the host rather than the simplified system of the Petri dish5.

The rapid development, small size and transparency of zebrafish are tremendous advantages for the study of innate immune control of infection1-4. Here we demonstrate techniques for infecting zebrafish larvae using the fungal pathogen Candida albicans by microinjection, methodology recently used to implicate phagocyte NADPH oxidase activity in control of fungal dimorphism5.

Disseminated candidiasis caused by the pathogen Candida albicans is a clinically important problem in hospitalized individuals and is associated with a 30 ...

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.

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 study of bacterial virulence often requires a suitable animal model. Mammalian models of infection are costly and may raise ethical issues. The use 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 ...

Analysis of the Epithelial Damage Produced by Entamoeba histolytica Infection

Entamoeba histolytica is the causative agent of human amoebiasis, a major cause of diarrhea and hepatic abscess in tropical countries. Infection is initiated by interaction of the pathogen with intestinal epithelial cells. This interaction leads to disruption of intercellular structures such as tight junctions (TJ). TJ ensure sealing of the epithelial layer to separate host tissue from gut lumen. Recent studies provide evidence that disruption of TJ by the parasitic protein EhCPADH112 is a prerequisite for E. histolytica invasion that is accompanied by epithelial barrier dysfunction. Thus, the analysis of molecular mechanisms involved in TJ disassembly during E. histolytica invasion is of paramount importance to improve our understanding of amoebiasis pathogenesis. This article presents an easy model that allows the assessment of initial host-pathogen interactions and the parasite invasion potential. Parameters to be analyzed include transepithelial electrical resistance, interaction of EhCPADH112 with epithelial surface receptors, changes in expression and localization of epithelial junctional markers and localization of parasite molecules within epithelial cells.

Analysis of the Epithelial Damage Produced by Entamoeba histolytica Infection

Human infection by Entamoeba histolytica leads to amoebiasis, a major cause of diarrhea in tropical countries. Infection is initiated by pathogen interactions with intestinal epithelial cells, provoking the opening of cell-cell contacts and consequently diarrhea, sometimes followed by liver infection. This article provides a model to assess the early host-pathogen interactions to improve our understanding of amoebiasis pathogenesis.

Entamoeba histolytica is the causative agent of human amoebiasis, a major cause of diarrhea and hepatic abscess in tropical countries. Infection is ...

Modeling Mucosal Candidiasis in Larval Zebrafish by Swimbladder Injection

Early defense against mucosal pathogens consists of both an epithelial barrier and innate immune cells. The immunocompetency of both, and their intercommunication, are paramount for the protection against infections. The interactions of epithelial and innate immune cells with a pathogen are best investigated in vivo, where complex behavior unfolds over time and space. However, existing models do not allow for easy spatio-temporal imaging of the battle with pathogens at the mucosal level.
The model developed here creates a mucosal infection by direct injection of the fungal pathogen, Candida albicans, into the swimbladder of juvenile zebrafish. The resulting infection enables high-resolution imaging of epithelial and innate immune cell behavior throughout the development of mucosal disease. The versatility of this method allows for interrogation of the host to probe the detailed sequence of immune events leading to phagocyte recruitment and to examine the roles of particular cell types and molecular pathways in protection. In addition, the behavior of the pathogen as a function of immune attack can be imaged simultaneously by using fluorescent protein-expressing C. albicans. Increased spatial resolution of the host-pathogen interaction is also possible using the described rapid swimbladder dissection technique.
The mucosal infection model described here is straightforward and highly reproducible, making it a valuable tool for the study of mucosal candidiasis. This system may also be broadly translatable to other mucosal pathogens such as mycobacterial, bacterial or viral microbes that normally infect through epithelial surfaces.

In vivo spatio-temporal interactions of pathogen and immune defenses at the mucosal level are not easily imaged in existing vertebrate hosts. The method presented here describes a versatile platform to study mucosal candidiasis in live vertebrates using the swimbladder of the juvenile zebrafish as an infection site.

Early defense against mucosal pathogens consists of both an epithelial barrier and innate immune cells. The immunocompetency of both, and their ...

Development of an in vitro model system for studying the interaction of Equus caballus IgE with its high-affinity receptor Fc&#949;RI

The interaction of IgE with its high-affinity Fc receptor (Fc&#949;RI) followed by an antigenic challenge is the principal pathway in IgE mediated allergic reactions. As a consequence of the high affinity binding between IgE and Fc&#949;RI, along with the continuous production of IgE by B cells, allergies usually persist throughout life, with currently no permanent cure available. Horses, especially race horses, which are commonly inbred, are a species of mammals that are very prone to the development of hypersensitivity responses, which can seriously affect their performance. Physiological responses to allergic sensitization in horses mirror that observed in humans and dogs. In this paper we describe the development of an in situ assay system for the quantitative assessment of the release of mediators of the allergic response pertaining to the equine system. To this end, the gene encoding equine Fc&#949;RI&#945; was transfected into and expressed onto the surface of parental Rat Basophil Leukemia (RBL-2H3.1) cells. The gene product of the transfected equine &#945;-chain formed a functional receptor complex with the endogenous rat &#946;- and &#947;-chains 1. The resultant assay system facilitated an assessment of the quantity of mediator secreted from equine Fc&#949;RI&#945; transfected RBL-2H3.1 cells following sensitization with equine IgE and antigenic challenge using &#946;-hexosaminidase release as a readout 2, 3. Mediator release peaked at 36.68% &#177; 4.88% at 100 ng ml-1 of antigen. This assay was modified from previous assays used to study human and canine allergic responses 4, 5. We have also shown that this type of assay system has multiple applications for the development of diagnostic tools and the safety assessment of potential therapeutic intervention strategies in allergic disease 6, 2, 3.

The current study describes the development and applications of a genetically engineered assay system based on the transfection of rat basophilic leukemia cells with the equine Fc&#949;RI&#945; gene. Transfected cells express a functional receptor where the release of mediators of the allergic response can be activated by IgE and antigen.

The interaction of IgE with its high-affinity Fc receptor (Fc&#949;RI) followed by an antigenic challenge is the principal pathway in IgE mediated ...

Fluorescence-based Neuraminidase Inhibition Assay to Assess the Susceptibility of Influenza Viruses to The Neuraminidase Inhibitor Class of Antivirals

The neuraminidase (NA) inhibitors are the only class of antivirals approved for the treatment and prophylaxis of influenza that are effective against currently circulating strains. In addition to their use in treating seasonal influenza, the NA inhibitors have been stockpiled by a number of countries for use in the event of a pandemic. It is therefore important to monitor the susceptibility of circulating influenza viruses to this class of antivirals. There are different types of assays that can be used to assess the susceptibility of influenza viruses to the NA inhibitors, but the enzyme inhibition assays using either a fluorescent substrate or a chemiluminescent substrate are the most widely used and recommended. This protocol describes the use of a fluorescence-based assay to assess influenza virus susceptibility to NA inhibitors. The assay is based on the NA enzyme cleaving the 2&#8242;-(4-Methylumbelliferyl)-&#945;-D-N-acetylneuraminic acid (MUNANA) substrate to release the fluorescent product 4-methylumbelliferone (4-MU). Therefore, the inhibitory effect of an NA inhibitor on the influenza virus NA is determined based on the concentration of the NA inhibitor that is required to reduce 50% of the NA activity, given as an IC50 value.

We describe the use of a phenotypic fluorescence-based neuraminidase inhibition assay to assess the susceptibility of influenza A and B viruses to the neuraminidase inhibitor class of antivirals.

The neuraminidase (NA) inhibitors are the only class of antivirals approved for the treatment and prophylaxis of influenza that are effective against ...

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.

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.

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

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 with Mycobacterium tuberculosis. Despite decades of research, many of the mechanisms behind the success of M. tuberculosis as a pathogenic organism remain to be investigated, and the development of safer, more effective antimycobacterial drugs are urgently needed to tackle the rise and spread of drug resistant tuberculosis. However, the progression of tuberculosis research is bottlenecked by traditional mammalian infection models that are expensive, time consuming, and ethically challenging. Previously we established the larvae of the insect Galleria mellonella (greater wax moth) as a novel, reproducible, low cost, high-throughput and ethically acceptable infection model for members of the M. tuberculosis complex. Here we describe the maintenance, preparation, and infection of G. mellonella with bioluminescent Mycobacterium bovis BCG lux. Using this infection model, mycobacterial dose dependent virulence can be observed, and a rapid readout of in vivo mycobacterial burden using bioluminescence measurements is easily achievable and reproducible. Although limitations exist, such as the lack of a fully annotated genome for transcriptomic analysis, ontological analysis against genetically similar insects can be carried out. As a low cost, rapid, and ethically acceptable model for tuberculosis, G. mellonella can be used as a pre-screen to determine drug efficacy and toxicity, and to determine comparative mycobacterial virulence prior to the use of conventional mammalian models. The use of the G. mellonella-mycobacteria model will lead to a reduction in the substantial number of animals currently used in tuberculosis research.

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

Galleria mellonella was recently established as a reproducible, cheap, and ethically acceptable infection model for the Mycobacterium tuberculosis complex. Here we describe and demonstrate the steps taken to establish successful infection of G. mellonella with bioluminescent Mycobacterium bovis BCG lux.

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 ...