We are grateful to Winnie Zhao and Yin Chen Wan for providing helpful comments on the manuscript. This work was supported by the Natural Sciences and Engineering Research Council of Canada (Grant #522691522691).

The present study uses wild-type C. elegans Bristol strain N2 grown at 21 &#176;C.
1. Preparation of media
<ol>
	<li>Prepare M9 media as per previous report21,22.</li>
	<li>Prepare nematode growth medium (NGM) as per previous report21,22. Pour 12 mL of NGM per 6 cm plate or 30 mL per 10 cm plate.</li>
	<li>Prepare Escherichia coli strain OP50-1 as per a previous report23.</li>
	<li>Prepare OP50-1-seeded NGM plates following the steps below.
	<ol>
		<li>Resuspend 10x concentrated OP50-1 by vortexing.</li>
		<li>Use a repeater pipette to seed the plates in the center. For 6 cm and 10 cm plates, seed with a volume of 200 &#181;L or 500 &#181;L, respectively.</li>
		<li>For 10 cm plates, use a glass spreader to spread the OP50-1 and create a lawn of bacteria. Do not spread the bacteria to the very edge of the plate. 
		NOTE: Ethanol dip and flame the spreader every five plates to ensure sterility. Allow the spreader to cool for a few seconds before spreading to prevent killing bacteria.</li>
		<li>Leave plates at room temperature for 2 days to dry and to ensure the growth of the bacterial lawn. 
		&#8203;NOTE: Seeded plates can be stored at room temperature for 2-3 weeks or at 4 &#176;C for up to 1 month.</li>
	</ol>
	</li>
</ol>
2. Maintenance of&#160;&#160;C. elegans
<ol>
	<li>Maintain worms on a constant bacterial food source on OP50-1-seeded NGM plates. 
	NOTE: Starvation may result in intergenerational phenotypes, which can affect results. A 10 cm OP50-1-seeded plate supports 2,500 L1s (the earliest larval stage of C. elegans) for up to 72 h.</li>
	<li>If the worms starve, grow for three generations on OP50-1 before using the worms for experiments.</li>
</ol>
3. Synchronization of&#160;&#160;C. elegans populations using sodium hypochlorite (bleaching)
NOTE: This step is very time-sensitive, so ensure the centrifuge is available before beginning. Alternative, less rapid bleaching protocols are available in the literature and may be used if preferred. To prevent 6% sodium hypochlorite from losing activity over time, store the reagent in the dark at 4 &#176;C and keep it for up to 1 year.
<ol>
	<li>Prepare 2 mL of bleach solution for each sample by mixing 400 &#181;L of 1 M NaOH, 250 &#181;L of 6% sodium hypochlorite (see Table of Materials), and 1350 &#181;L of distilled water.</li>
	<li>Wash adult worms (~72 h post-hatch) off the plates into a microcentrifuge tube using 1 mL of M9 media. 
	NOTE: If many worms remain on the plates, pellet the worms by centrifugation at 1,400 x g for 30 s at room temperature, remove the supernatant using a 1 mL pipette tip, and perform additional plate washes.</li>
	<li>Centrifuge at 1,400 x g for 30 s at room temperature to pellet the worms, then wash 2x with 1 mL of M9 media to remove bacteria.</li>
	<li>Remove the supernatant, add 1 mL of the prepared bleach solution (Step 3.1.), and set a timer for 1 min. Invert the tubes 3-4x. 
	NOTE: Under a light microscope, the worms can be seen to stop thrashing.</li>
	<li>After 1 min, centrifuge at 3,000 x g for 30 s at room temperature to pellet the worms, then remove the supernatant and add 1 mL of fresh bleach solution.</li>
	<li>Monitor the tube(s) closely under a light microscope to visualize the worm carcasses splitting open and releasing embryos. Immediately advance to the next step when ~50%-80% of the carcasses have split open and many embryos have been released. 
	NOTE: Depending on the number of animals and the worm strain, this step can take 15 s to 4 min. For unknown reasons, N. parisii-infected worms often take longer to bleach than uninfected animals.</li>
	<li>Centrifuge at 3,000 x g for 30 s at room temperature to pellet the embryos, then wash 3x in 1 mL of M9 media. 
	NOTE: Washes dilute the residual hypochlorite solution from the tubes and must be performed quickly to ensure the bleach solution does not damage the embryos.</li>
	<li>Add 1 mL of M9 media to the final pellet and transfer to a 15 mL conical tube containing 4 mL of M9 media. Suspend the embryos in a final volume of 5 mL.</li>
	<li>Spin the tubes on a mechanical rotor (see Table of Materials) at 21 &#176;C for 18-24 h to hatch the embryos into L1s. 
	NOTE: As N. parisii infects the worm intestine and does not invade the germline, infected parents do not pass the infection to their progeny by vertical transmission19. Bleaching of infected adults synchronizes F1 animals and destroys N. parisii spores, ensuring F1 progeny are not infected by spores carried over from the parents.</li>
	<li>Centrifuge the tubes for 1 min at 1,800 x g at room temperature to pellet the L1s, and discard all but 1 mL of the supernatant.</li>
	<li>Pipette 1-5 &#181;L of L1 mixture onto an unseeded NGM plate, and immediately count the number of L1s in the droplet by visualizing under a light microscope. Repeat 3x and average the counts to determine the concentration and total number of L1s. 
	&#8203;NOTE: The majority of the embryos should be hatched, and the L1s should be rapidly moving in the liquid droplet. If a large proportion of unhatched embryos and lethargic (slow-moving) L1s remain, this indicates bleaching for too long.</li>
</ol>
4. Preparation of&#160;&#160;N. parisii spores
<ol>
	<li>Prepare N. parisii spores following previously published references19,24.</li>
</ol>
5. Infection of&#160;&#160;C. elegans with&#160;&#160;N. parisii to yield immune-primed offspring
<ol>
	<li>One day before planned infections, move the required number of 10 cm unseeded NGM plates from 4 &#176;C to room temperature.</li>
	<li>Immediately before infection, centrifuge ~2,500 synchronized L1 worms at 1,400 x g for 30 s at room temperature in a microcentrifuge tube to pellet the worms. Remove supernatant with a pipette tip to leave worms in ~50 &#181;L of M9 media.</li>
	<li>Add 1 mL of 10x OP50-1 to the L1s and N. parisii spores to the desired concentration (typically ~2.5 million spores). As an uninfected control, prepare an equivalent tube of L1s and OP50-1 with a volume of M9 media equivalent to the volume of spore preparation used. 
	NOTE: To obtain immune-primed offspring, use a spore dose high enough so that &#62;90% of the animals are infected by 72 h (as measured by DY96 staining, Step 7) but low enough so that &#62;80% of animals are still gravid. This ensures that most offspring come from infected parents and that bleaching of the parents yields sufficient embryos for F1 testing. Though spore preparations vary in concentration and infectivity, a suitable parental dose is typically between 5-15 &#181;L of spore preparation (~2.5 million spores) per 10 cm plate.</li>
	<li>Vortex briefly to mix and plate the above 1 mL of worms onto a 10 cm unseeded NGM plate. Swirl to ensure the liquid spreads over the whole plate.</li>
	<li>Dry the plates in a clean cabinet with the lids off for 10-20 min or until completely dry, before incubating at 21 &#176;C for 72 h.</li>
	<li>At 72 h post-infection (hpi), wash the worms off the plates into a microcentrifuge tube using 1 mL of M9 media. If many worms remain on the plates, pellet the worms by centrifugation at 1,400 x g for 30 s, remove the supernatant, and perform additional plate washes.</li>
	<li>Centrifuge at 1,400 x g for 30 s at room temperature to pellet worms, wash 2x with 1 mL of M9 media or until the supernatant is clear. Resuspend in a final volume of 1 mL of M9 media.</li>
	<li>Pipette up and down to mix, then transfer 100 &#181;L of the suspended worms to a fresh tube. 
	NOTE: This sample of ~250 adults can later be fixed and stained to determine parental worm fitness and infection status, as described in Step 7 and the beginning of Step 3.</li>
	<li>To break open the adult worms and release F1 embryos for testing, bleach the remaining 900 &#181;L of suspended worms, as described in Step 3. 
	&#8203;NOTE: This step also destroys any N. parisii spores before subsequent infection assays.</li>
</ol>
6. Testing inherited immunity to&#160;&#160;N. parisii in&#160;&#160;C. elegans
<ol>
	<li>Perform infections as described in Steps 5.1.-5.6., with modifications as mentioned below. 
	NOTE: Infection assays on F1s can be scaled down and performed on 6 cm NGM plates using ~1,000 L1 worms and 400 &#181;L of 10x OP50-1. A &#34;high&#34; dose of spores must also be used, such that ~100% of na&#239;ve F1s (i.e., worms from uninfected parents) are infected, and only ~10% of na&#239;ve F1s are gravid. Though spore preparations vary in concentration and infectivity, a suitable dose is typically between 5-15 &#181;L (~2.5 million spores) per 6 cm plate.</li>
	<li>At 72 hpi, fix and stain to determine worm fitness and infection status, as described in Step 7.</li>
</ol>
7. DY96 staining of&#160;&#160;C. elegans to visualize embryos and microsporidia spores
NOTE: DY96 is a green fluorescent chitin-binding dye that stains worm embryos and microsporidia spore walls19,15,25. This allows simultaneous monitoring of the fitness and infection status of the worms.
<ol>
	<li>Wash the worms off the plates into a microcentrifuge tube using 1 mL of M9 media containing 0.1% Tween-20. If many worms remain on the plates, pellet the worms by centrifugation at 1,400 x g for 30 s at room temperature, remove the supernatant using a pipette tip, and perform additional plate washes.</li>
	<li>Centrifuge at 1,400 x g for 30 s at room temperature to pellet the worms, and wash 2x with 1 mL of M9 media containing 0.1% Tween-20 or until the supernatant is clear.</li>
	<li>Remove the supernatant, add 700 &#181;L of acetone and leave the worms to fix at room temperature for 10 min. 
	NOTE: At this point, the worms can be stored at -20 &#176;C for processing up to several months later, if desired.</li>
	<li>Centrifuge at 10,000 x g for 30 s at room temperature to pellet the fixed worms, and wash 2x with 1 mL of PBS containing 0.1% Tween-20 (PBST).</li>
	<li>Prepare 50 mL of DY96 working solution: PBST, 0.1% (v/v) sodium dodecyl sulfate (SDS), and 20 &#181;g/mL DY96 (from a 5 mg/mL stock solution in distilled water) (see Table of Materials). Wrap the tube in foil and store it in a drawer to prevent exposure to light. 
	NOTE: DY96 stock and working solutions can be stored for &#62;1 year at room temperature if kept in the dark.</li>
	<li>Centrifuge at 10,000 x g for 30 s at room temperature to pellet the worms and remove supernatant. Add 500 &#181;L of DY96 working solution to the pellet and rotate for 30 min at room temperature.</li>
	<li>Centrifuge at 10,000 x g for 30 s at room temperature to pellet the worms. Remove the supernatant and add 15 &#181;L of the mounting medium with/without DAPI (see Table of Materials).</li>
	<li>Pipette 10 &#181;L of the solution containing stained worms onto a microscope slide and place a coverslip over the top. 
	NOTE: Slides and additional samples can be stored at 4 &#176;C in the dark for long-term storage.</li>
</ol>
8. Imaging and analysis of DY96-stained worms to assess worm fitness and infection status
<ol>
	<li>To analyze the DY96-stained worms (mounted on slides, as in Step 7.8.), perform imaging using the GFP channel of a fluorescence microscope.</li>
	<li>To determine the fitness of the worm populations, use a 5x or 10x objective to assay the gravidity of &#62;100 worms per condition. 
	NOTE: Worms carrying &#8805;1 embryo are considered gravid. Mechanical cell counters can help to minimize human error when counting. Worm embryos are less stained (less fluorescent) than microsporidia spores19. Embryos are ovoid, ~50 &#181;m x ~30 &#181;m, and found in the midbody of healthy C. elegans adults. Healthy, uninfected adults carry 10-20 embryos organized in rows along their body length26.</li>
	<li>To reveal more subtle differences in fitness, perform embryo counts. For this, manually count the number of embryos in 20-30 individual worms per condition.</li>
	<li>To determine how infected the worm populations are, use a 5x-40x objective to assess the infection status of &#62;100 worms per condition.Worms comprising any number of intracellular gut spores are considered infected. 
	NOTE: Microsporidia spores are brighter than worm embryos. The bean-shaped spores of N. parisii are ~2.2 &#181;m x ~0.8 &#181;m and are produced in the worm&#39;s intestinal cells from ~48 hpi15. Worms in which spores are seen exclusively in the gut lumen and not along the intestinal walls are not considered infected19. This is because these spores likely represent newly ingested spores that do not result from infection of the individual.</li>
	<li>Using image analysis software (see Table of Materials), quantify parasite burden and worm size following the steps below.
	<ol>
		<li>Image 20-30 worms mounted on microscope slides using a fluorescent upright stereoscope for each sample. Capture images in Brightfield, DAPI, and GFP channels. Choose an appropriate exposure in the GFP channel such that the fluorescence in the most infected samples is not saturated. 
		NOTE: The infection can still be visualized in the least infected samples. Images are typically taken using a 5x objective.</li>
		<li>Open files in FIJI/ImageJ. Depending on the file type, the Bio-Formats Importer plugin may be required to open images in ImageJ.</li>
		<li>Outline 20-30 individual worms in images using brightfield or DAPI images and the Polygon selections tool in the toolbar. Save each outline with Analyze &#62; Tools &#62; Regions of Interest (ROI) Manager in the dropdown menu by clicking on Add [t]. Outline animals that are fully visible in the frame. 
		NOTE: Worm outlines can be revisited later by saving the file with outlines as a Tiff.</li>
		<li>Click on Deselect in the ROI manager to remove all outlines from the image and click on Image &#62; Duplicate in the dropdown menu. Type the number corresponding to the channel of interest into the &#34;Channels&#34; box to duplicate the GFP channel for parasite burden (e.g., 2).</li>
		<li>Threshold this channel using Image &#62; Adjust &#62; Threshold to a level at which the bright parasite burden is captured but the dimmer fluorescence from worm embryos is not, and then click on Apply. Take note of the threshold values applied and use them consistently for all images of the same experiment. 
		NOTE: Check the threshold is an appropriate value for both the least and most infected samples before analyzing all the images.</li>
		<li>Select Analyze &#62; Set measurements and check the box for Area Fraction. Select single worm outlines in turn from the ROI manager and click on the Analyze &#62; Measure function. A Results window will provide the measurement for %Area (i.e., % parasite burden). 
		NOTE: If the fluorescence from the embryos is so bright that it crosses the threshold, the Paintbrush tool in black in the toolbar can be used to manually erase these regions prior to measuring %Area.</li>
		<li>To determine worm size as a readout of fitness, outline worms as described in Step 8.3. Select Analyze &#62; Set measurements and check the box for Area. Select single worm outlines in turn from the ROI manager and click on the Analyze &#62; Measure function. A Results window will provide the measurement for %Area.</li>
	</ol>
	</li>
</ol>
9. FISH assay to assess invasion of&#160;&#160;C. elegans by microsporidia and spore firing
NOTE: The MicroB FISH probe recognizes a conserved region of microsporidian 18s rRNA and can be used to label intracellular sporoplasms (i.e., invaded host cells) and the genetic material within spores.
<ol>
	<li>Infect 6,000 bleach synchronized L1 worms with 4 million spores of N. parisii and&#160;10 &#181;L of 10x OP50-1 in a final volume of 400 &#181;L that is made up with M9 media.</li>
	<li>Plate the mixture on a 6 cm unseeded NGM plate, dry in a clean cabinet for 10-20 min, and place at 21 &#176;C.</li>
	<li>After 30 min, wash the animals off the plates with 1 mL of M9 media containing 0.1% Tween-20.</li>
	<li>Pellet the worms by centrifuging at 1,400 x g for 1 min at room temperature.</li>
	<li>Remove the supernatant using a pipette tip, add 700 &#181;L of acetone, and allow it to sit for 10 min at room temperature. 
	NOTE: At this point, the worms can be stored at -20 &#176;C for processing at a later time.</li>
	<li>Perform overnight FISH assay using the MicroB probe following previously published reports27,19. 
	NOTE: To assess spore firing, supplement the final 500 &#181;L of wash buffer with 20 &#181;g/mL of DY96 to the samples and rotate for 30 min at 21 &#176;C before continuing with the protocol as normal.</li>
	<li>Store the slides in a slide box at 4 &#176;C. For long-term storage (i.e., several months), store additional samples in microcentrifuge tubes at 4 &#176;C in darkness.</li>
	<li>To assess invasion in FISH-stained worms, look at the worms using the red channel of a fluorescence microscope. Visualize infection events at high magnification (63x objective) as L1 animals are small, and sporoplasms will be in the early stages of replication. To assess spore firing, use a 63x objective and both red and green fluorescence channels. 
	NOTE: Spores in which GFP and mCherry signal colocalize are considered unfired; empty GFP spore cases are considered fired.</li>
	<li>To assay invasion or spore firing, manually count the number of sporoplasms (mCherry foci in the intestinal cells) or the percentage of spores fired in 20-30 worms per condition. Adjust the focus in the z-plane to capture all infection events and spores, which are often found in different planes. 
	NOTE: Sporoplasms are found exclusively within the intestinal cells. The highest concentration of sporoplasms is typically found immediately after the pharynx. If samples are also stained with DY96, look for the presence of sporoplasms that do not colocalize with the spore.</li>
</ol>

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C., Troemel, E. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Non-lytic,+actin-based+exit+of+intracellular+parasites+from+C.+elegans+intestinal+cells.">Non-lytic, actin-based exit of intracellular parasites from C. elegans intestinal cells.</a> PLOS Pathogens. 7, 1002227 (2011).</li><li>Botts, M. R., Cohen, L. B., Probert, C. S., Wu, F., Troemel, E. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microsporidia+intracellular+development+relies+on+myc+interaction+network+transcription+factors+in+the+host.">Microsporidia intracellular development relies on myc interaction network transcription factors in the host.</a> G3 Genes|Genomes|Genetics. 6 (9), 2707-2716 (2016).</li><li>Corsi, A. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Transparent+window+into+biology:+A+primer+on+Caenorhabditis+elegans.">A Transparent window into biology: A primer on Caenorhabditis elegans.</a> WormBook. , 1-31 (2015).</li><li>Rivera, D. E., La&#382;eti&#263;, V., Troemel, E. R., Luallen, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RNA+fluorescence+in+situ+hybridization+(FISH)+to+visualize+microbial+colonization+and+infection+in+the+Caenorhabditis+elegans+intestines.">RNA fluorescence in situ hybridization (FISH) to visualize microbial colonization and infection in the Caenorhabditis elegans intestines.</a> bioRxiv. , (2022).</li><li>Zhang, G., 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=A+large+collection+of+novel+nematode-infecting+microsporidia+and+their+diverse+interactions+with+Caenorhabditis+elegans+and+other+related+nematodes.">A large collection of novel nematode-infecting microsporidia and their diverse interactions with Caenorhabditis elegans and other related nematodes.</a> PLoS Pathogens. 12, 1006093 (2016).</li><li>Luallen, R. J., 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=Discovery+of+a+natural+microsporidian+pathogen+with+a+broad+tissue+tropism+in+Caenorhabditis+elegans.">Discovery of a natural microsporidian pathogen with a broad tissue tropism in Caenorhabditis elegans.</a> PLoS Pathogens. 12, 1005724 (2016).</li><li>Troemel, E. R., F&#233;lix, M. -. A., Whiteman, N. K., Barri&#232;re, A., Ausubel, F. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microsporidia+are+natural+intracellular+parasites+of+the+nematode+Caenorhabditis+elegans.">Microsporidia are natural intracellular parasites of the nematode Caenorhabditis elegans.</a> PLoS Biology. 6, 309 (2008).</li><li>Burton, N. O., 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=Intergenerational+adaptations+to+stress+are+evolutionarily+conserved,+stress-specific,+and+have+deleterious+trade-offs.">Intergenerational adaptations to stress are evolutionarily conserved, stress-specific, and have deleterious trade-offs.</a> eLife. 10, 73425 (2021).</li><li>Jaroenlak, 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=3-Dimensional+organization+and+dynamics+of+the+microsporidian+polar+tube+invasion+machinery.">3-Dimensional organization and dynamics of the microsporidian polar tube invasion machinery.</a> PLoS Pathogens. 16, 1008738 (2020).</li><li>Weidner, E., Manale, S. B., Halonen, S. K., Lynn, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protein-membrane+interaction+is+essential+to+normal+assembly+of+the+microsporidian+spore+invasion+tube.">Protein-membrane interaction is essential to normal assembly of the microsporidian spore invasion tube.</a> The Biological Bulletin. 188 (2), 128-135 (1995).</li></ol>

The authors have nothing to disclose.

The present protocol describes the study of microsporidia and inherited immunity in a simple and genetically tractable N. parisii -C. elegans infection model.
Spore preparation is an intensive protocol that typically yields enough spores for 6 months of experiments, depending on productivity24. Importantly, infectivity must be determined for each new spore &#34;lot&#34; before using it for the experiments. Due to the variability in infectivity between ...

Inherited immunity is an epigenetic phenomenon whereby parental exposure to pathogens can enable the production of infection-resistant offspring. This type of immune memory has been shown in many invertebrates that lack adaptive immune systems and can protect against viral, bacterial, and fungal disease1. While inherited immunity has important implications for understanding both health and evolution, the molecular mechanisms underlying this protection are largely unknown. This is partly because many of the animals in which inherited immunity has been described are not established model organisms for research. In contrast, studies in the transparent nematode Caenorhabditis elegans benefit from an extensive genetic and biochemical toolkit2,3, a highly annotated genome4,5, and a short generation time. Indeed, research in C. elegans has enabled fundamental advances in the fields of epigenetics and innate immunity6,7, and it is now an established model for studying immune memory8,9.
Microsporidia are fungal pathogens that infect almost all animals and cause lethal infections in immunocompromised humans10. Infection begins when a microsporidia spore injects or &#34;fires&#34; its cellular contents (sporoplasm) into a host cell using a structure called a polar tube. Intracellular replication of the parasite results in the formation of meronts, which ultimately differentiate into mature spores that can exit the cell11,12. While these parasites are detrimental to both human health and food security, there is much still to learn about their infection biology12. Nematocida parisii is a natural microsporidian parasite that replicates exclusively in the intestinal cells of worms, resulting in reduced fecundity and, ultimately, death. The N. parisii -C. elegans infection model has been used to show: (1) the role of autophagy in pathogen clearance13, (2) how microsporidia can exit infected cells non-lytically14, (3) how pathogens can spread from cell-to-cell by forming syncytia15, (4) the proteins N. parisii use to interface with its host16, and (5) the regulation of the transcriptional intracellular pathogen response (IPR)17,18.
Protocols for the infection of C. elegans are described in the current work and can be used to reveal the unique microsporidia biology and dissect the host&#39;s response to infection. The microscopy of fixed worms stained with the chitin-binding dye Direct Yellow 96 (DY96) shows the infection spread of chitin-containing microsporidia spores throughout the intestine. DY96 staining also enables the visualization of chitin-containing worm embryos for the simultaneous assessment of worm gravidity (ability to produce embryos) as a readout of host fitness.
Recent work has revealed that C. elegans infected with N. parisii produce offspring that are robustly resistant to the same infection19. This inherited immunity lasts a single generation and is dose-dependent, as offspring from more heavily infected parents are more resistant to microsporidia. Interestingly, N. parisii-primed offspring are also more resistant to the bacterial gut pathogen Pseudomonas aeruginosa, though they are not protected against the natural pathogen Orsay virus19. The present work also shows that immune-primed offspring limit host cell invasion by microsporidia. The method also describes the collection of immune-primed offspring and how FISH can be used to detect N. parisii RNA in intestinal cells to assay host cell invasion and spore firing20.
Together, these protocols provide a solid foundation for studying microsporidia and inherited immunity in C. elegans. It is hoped that future work in this model system will enable important discoveries in the nascent field of inherited immunity. These techniques are also likely to be starting points for investigating microsporidia-induced inherited immunity in other host organisms.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>2.0 mm zirconia beads</td>
			<td>Biospec Products Inc.</td>
			<td>11079124ZX</td>
			<td></td>
		</tr>
		<tr>
			<td>10 mL syringe</td>
			<td>Fisher Scientific</td>
			<td>1482613</td>
			<td></td>
		</tr>
		<tr>
			<td>5 &#956;m filter</td>
			<td>Millipore Sigma</td>
			<td>SLSV025LS</td>
			<td></td>
		</tr>
		<tr>
			<td>Axio Imager 2</td>
			<td>Zeiss</td>
			<td>-</td>
			<td>Fluorescent microscope for imaging of DY96- and FISH- stained worms on microscope slides</td>
		</tr>
		<tr>
			<td>Axio Zoom V.16 Fluorescence Stereo Zoom Microscope</td>
			<td>Zeiss</td>
			<td>-</td>
			<td>For live imaging of fluorescent transgenic animals to visualize the IPR</td>
		</tr>
		<tr>
			<td>Baked EdgeGARD Horizontal Flow Clean Bench</td>
			<td>Baker</td>
			<td>-</td>
			<td></td>
		</tr>
		<tr>
			<td>Bead disruptor, Genie SI-D238 Analog Disruptor Genie Cell Disruptor, 120 V</td>
			<td>Global Industrial</td>
			<td>T9FB893150</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell-VU slide, Millennium Sciences Disposable Sperm Count Cytometers</td>
			<td>Fisher Scientific</td>
			<td>DRM600</td>
			<td></td>
		</tr>
		<tr>
			<td>Direct Yellow 96</td>
			<td>Sigma-Aldrich</td>
			<td>S472409-1G</td>
			<td></td>
		</tr>
		<tr>
			<td>EverBrite Mounting Medium with DAPI</td>
			<td>Biotium</td>
			<td>23001</td>
			<td></td>
		</tr>
		<tr>
			<td>EverBrite Mounting Medium without DAPI</td>
			<td>Biotium</td>
			<td>23002</td>
			<td></td>
		</tr>
		<tr>
			<td>Fiji/ImageJ software</td>
			<td>ImageJ</td>
			<td>https://imagej.net/software/fiji/downloads</td>
			<td></td>
		</tr>
		<tr>
			<td>Mechanical rotor</td>
			<td>Thermo Sceintific</td>
			<td>415110 / 1834090806873</td>
			<td>Used to spin tubes of bleached embryos for overnight hatching</td>
		</tr>
		<tr>
			<td>MicroB FISH probe</td>
			<td>Biosearch Technologies Inc.</td>
			<td>-</td>
			<td>Synthesized with a Quasar 570 (Cy3) 5&#39; modification and HPLC purified, CTCTCGGCACTCCTTCCTG</td>
		</tr>
		<tr>
			<td>N2</td>
			<td>Wild-type, Bristol strain</td>
			<td>Default strain</td>
			<td>Caenorhabditis Genetics Center (CGC)</td>
		</tr>
		<tr>
			<td>Sodium dodecyl sulfate (SDS)</td>
			<td>Sigma-Aldrich</td>
			<td>L3771-100G</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium hydroxide solution (5 N)</td>
			<td>Fisher Chemical</td>
			<td>FLSS256500</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium hypochlorite solution (6%)</td>
			<td>Fisher Chemical</td>
			<td>SS290-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Stemi 508 Stereo Microscope</td>
			<td>Zeiss</td>
			<td>-</td>
			<td>For daily maintenance of worms and counting of L1 worms for assay set ups</td>
		</tr>
		<tr>
			<td>Tween-20</td>
			<td>Sigma-Aldrich</td>
			<td>P1379-100ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Vectashield + A16</td>
			<td>Biolynx</td>
			<td>VECTH1500</td>
			<td></td>
		</tr>
	</tbody>
</table>

studying inherited immunity in a caenorhabditis elegans model of microsporidia infection

Inherited immunity describes how some animals can pass on the "memory" of a previous infection to their offspring. This can boost pathogen resistance in their progeny and promote survival. While inherited immunity has been reported in many invertebrates, the mechanisms underlying this epigenetic phenomenon are largely unknown. The infection of Caenorhabditis elegans by the natural microsporidian pathogen Nematocida parisii results in the worms producing offspring that are robustly resistant to microsporidia. The present protocol describes the study of intergenerational immunity in the simple and genetically tractable N. parisii -C. elegans infection model. The current article describes methods for infecting C. elegans and generating immune-primed offspring. Methods are also given for assaying resistance to microsporidia infection by staining for microsporidia and visualizing infection by microscopy. In particular, inherited immunity prevents host cell invasion by microsporidia, and fluorescence in situ hybridization (FISH) can be used to quantify invasion events. The relative amount of microsporidia spores produced in the immune-primed offspring can be quantified by staining the spores with a chitin-binding dye. To date, these methods have shed light on the kinetics and pathogen specificity of inherited immunity, as well as the molecular mechanisms underlying it. These techniques, alongside the extensive tools available for C. elegans research, will enable important discoveries in the field of inherited immunity.

In the present study, parental populations of C. elegans (P0) were infected at the L1 stage with a low dose of N. parisii spores. These infection conditions are typically used to obtain high numbers of microsporidia-resistant F1 progeny through bleaching of the parents. Infected parental populations and uninfected controls were fixed at 72 hpi and stained with DY96 to visualize the worm embryos and microsporidia spores (Figure 1A). Infected animals are small, contain many m...

Watch this Scientific Journal Video about Studying Inherited Immunity in a Caenorhabditis elegans Model of Microsporidia Infection at JoVE.com

Studying Inherited Immunity in a Caenorhabditis elegans Model of Microsporidia Infection

The infection of Caenorhabditis elegans by the microsporidian parasite Nematocida parisii enables the worms to produce offspring that are highly resistant to the same pathogen. This is an example of inherited immunity, a poorly understood epigenetic phenomenon. The present protocol describes the study of inherited immunity in a genetically tractable worm&#160;model.

Studying Inherited Immunity in a Caenorhabditis elegans Model of Microsporidia Infection

Inherited immunity describes how some animals can pass on the "memory" of a previous infection to their offspring. This can boost pathogen resistance in ...

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Immunology and Infection

2.2K Views.  University of Toronto. The infection of Caenorhabditis elegans by the microsporidian parasite Nematocida parisii enables the worms to produce offspring that are highly resistant to the same pathogen. This is an example of inherited immunity, a poorly understood epigenetic phenomenon. The present protocol describes the study of inherited immunity in a genetically tractable worm model.

Video: Studying Inherited Immunity in a Caenorhabditis elegans Model of Microsporidia Infection

Trichuris muris Infection: A Model of Type 2 Immunity and Inflammation in the Gut

Trichuris muris is a natural pathogen of mice and is biologically and antigenically similar to species of Trichuris that 
infect humans and livestock1. Infective eggs are given by oral gavage, hatch in the distal small intestine, invade the intestinal epithelial cells (IECs) 
that line the crypts of the cecum and proximal colon and upon maturation the worms release eggs into the environment1. This model is a powerful tool to 
examine factors that control CD4+ T helper (Th) cell activation as well as changes in the intestinal epithelium. The immune response that occurs in 
resistant inbred strains, such as C57BL/6 and BALB/c, is characterized by Th2 polarized cytokines (IL-4, IL-5 and IL-13) and expulsion of worms while Th1-associated 
cytokines (IL-12, IL-18, IFN-&gamma;) promote chronic infections in genetically susceptible AKR/J mice2-6. Th2 cytokines promote physiological changes in 
the intestinal microenvironment including rapid turnover of IECs, goblet cell differentiation, recruitment and changes in epithelial permeability and smooth muscle 
contraction, all of which have been implicated in worm expulsion7-15. Here we detail a protocol for propagating Trichuris muris eggs which can 
be used in subsequent experiments. We also provide a sample experimental harvest with suggestions for post-infection analysis. Overall, this protocol will provide 
researchers with the basic tools to perform a Trichuris muris mouse infection model which can be used to address questions pertaining to Th proclivity in 
the gastrointestinal tract as well as immune effector functions of IECs.

Trichuris muris Infection: A Model of Type 2 Immunity and Inflammation in the Gut

Trichuris muris infection is an intestinal model of Th2 immunity where resistant mice generate a protective Th2 response and susceptible mice generate a pathological Th1 response.

Trichuris muris is a natural pathogen of mice and is biologically and antigenically similar to species of Trichuris that 
infect humans and livestock1. ...

Selection of Plasmodium falciparum Parasites for Cytoadhesion to Human Brain Endothelial Cells

Most human malaria deaths are caused by blood-stage Plasmodium falciparum parasites. Cerebral malaria, the most life-threatening complication of the disease, is characterised by an accumulation of Plasmodium falciparum infected red blood cells (iRBC) at pigmented trophozoite stage in the microvasculature of the brain2-4. This microvessel obstruction (sequestration) leads to acidosis, hypoxia and harmful inflammatory cytokines (reviewed in 5). Sequestration is also found in most microvascular tissues of the human body2, 3. The mechanism by which iRBC attach to the blood vessel walls is still poorly understood.
The immortalized Human Brain microvascular Endothelial Cell line (HBEC-5i) has been used as an in vitro model of the blood-brain barrier6. However, Plasmodium falciparum iRBC attach only poorly to HBEC-5i in vitro, unlike the dense sequestration that occurs in cerebral malaria cases. We therefore developed a panning assay to select (enrich) various P. falciparum strains for adhesion to HBEC-5i in order to obtain populations of high-binding parasites, more representative of what occurs in vivo.
A sample of a parasite culture (mixture of iRBC and uninfected RBC) at the pigmented trophozoite stage is washed and incubated on a layer of HBEC-5i grown on a Petri dish. After incubation, the dish is gently washed free from uRBC and unbound iRBC. Fresh uRBC are added to the few iRBC attached to HBEC-5i and incubated overnight. As schizont stage parasites burst, merozoites reinvade RBC and these ring stage parasites are harvested the following day. Parasites are cultured until enough material is obtained (typically 2 to 4 weeks) and a new round of selection can be performed. Depending on the P. falciparum strain, 4 to 7 rounds of selection are needed in order to get a population where most parasites bind to HBEC-5i. The binding phenotype is progressively lost after a few weeks, indicating a switch in variant surface antigen gene expression, thus regular selection on HBEC-5i is required to maintain the phenotype.
In summary, we developed a selection assay rendering P. falciparum parasites a more "cerebral malaria adhesive" phenotype. We were able to select 3 out of 4 P. falciparum strains on HBEC-5i. This assay has also successfully been used to select parasites for binding to human dermal and pulmonary endothelial cells. Importantly, this method can be used to select tissue-specific parasite populations in order to identify candidate parasite ligands for binding to brain endothelium. Moreover, this assay can be used to screen for putative anti-sequestration drugs7.

Selection of Plasmodium falciparum Parasites for Cytoadhesion to Human Brain Endothelial Cells

An in vitro model for cerebral malaria sequestration is described1. Plasmodium falciparum infected red blood cells are selected for binding to immortalized human brain microvascular endothelial cells. The selected parasites show a distinct phenotype. The selection process can be applied using various P. falciparum strains and endothelial cell lines.

Most human malaria deaths are caused by blood-stage Plasmodium falciparum parasites. Cerebral malaria, the most life-threatening complication of the ...

A Flow Adhesion Assay to Study Leucocyte Recruitment to Human Hepatic Sinusoidal Endothelium Under Conditions of Shear Stress

Leucocyte infiltration into human liver tissue is a common process in all adult inflammatory liver diseases. Chronic infiltration can drive the development of fibrosis and progression to cirrhosis. Understanding the molecular mechanisms that mediate leucocyte recruitment to the liver could identify important therapeutic targets for liver disease. The key interaction during leucocyte recruitment is that of inflammatory cells with endothelium under conditions of shear stress. Recruitment to the liver occurs within the low shear channels of the hepatic sinusoids which are lined by hepatic sinusoidal endothelial cells (HSEC). The conditions within the hepatic sinusoids can be recapitulated by perfusing leucocytes through channels lined by human HSEC monolayers at specific flow rates. In these conditions leucocytes undergo a brief tethering step followed by activation and firm adhesion, followed by a crawling step and subsequent transmigration across the endothelial layer. Using phase contrast microscopy, each step of this &#39;adhesion cascade&#39; can be visualized and recorded followed by offline analysis. Endothelial cells or leucocytes can be pretreated with inhibitors to determine the role of specific molecules during this process.

Leucocyte recruitment to the liver occurs within the specialized channels of the hepatic sinusoids which are lined by unique hepatic sinusoidal endothelial cells. Phase contrast microscopy of leucocyte recruitment across human hepatic sinusoidal endothelium under conditions of physiological shear stress can facilitate the elucidation of the molecular mechanisms which underlie this process.

Leucocyte infiltration into human liver tissue is a common process in all adult inflammatory liver diseases. Chronic infiltration can drive the ...

High Yield Purification of Plasmodium falciparum Merozoites For Use in Opsonizing Antibody Assays

Plasmodium falciparum merozoite antigens are under development as potential malaria vaccines. One aspect of immunity against malaria is the removal of free merozoites from the blood by phagocytic cells. However assessing the functional efficacy of merozoite specific opsonizing antibodies is challenging due to the short half-life of merozoites and the variability of primary phagocytic cells. Described in detail herein is a method for generating viable merozoites using the E64 protease inhibitor, and an assay of merozoite opsonin-dependent phagocytosis using the pro-monocytic cell line THP-1. E64 prevents schizont rupture while allowing the development of merozoites which are released by filtration of treated schizonts. &#160;Ethidium bromide labelled merozoites are opsonized with human plasma samples and added to THP-1 cells. Phagocytosis is assessed by a standardized high throughput protocol. Viable merozoites are a valuable resource for assessing numerous aspects of P. falciparum biology, including assessment of immune function. Antibody levels measured by this assay are associated with clinical immunity to malaria in naturally exposed individuals. The assay may also be of use for assessing vaccine induced antibodies. &#160;

High Yield Purification of Plasmodium falciparum Merozoites For Use in Opsonizing Antibody Assays

Measuring antibody function is key to understanding immunity to Plasmodium falciparum malaria. This method describes the purification of viable merozoites, and measurement of opsonization-dependent phagocytosis by flow cytometry.

Plasmodium falciparum merozoite antigens are under development as potential malaria vaccines. One aspect of immunity against malaria is the removal of ...

A Protocol to Infect Caenorhabditis elegans with Salmonella typhimurium

In the last decade, C. elegans has emerged as an invertebrate organism to study interactions between hosts and pathogens, including the host defense against gram-negative bacterium Salmonella typhimurium. Salmonella establishes persistent infection in the intestine of C. elegans and results in early death of infected animals. A number of immunity mechanisms have been identified in C. elegans to defend against Salmonella infections. Autophagy, an evolutionarily conserved lysosomal degradation pathway, has been shown to limit the Salmonella replication in C. elegans and in mammals. Here, a protocol is described to infect C. elegans with Salmonella typhimurium, in which the worms are exposed to Salmonella for a limited time, similar to Salmonella infection in humans. Salmonella infection significantly shortens the lifespan of C. elegans. Using the essential autophagy gene bec-1 as an example, we combined this infection method with C. elegans RNAi feeding approach and showed this protocol can be used to examine the function of C. elegans host genes in defense against Salmonella infection. Since C. elegans whole genome RNAi libraries are available, this protocol makes it possible to comprehensively screen for C. elegans genes that protect against Salmonella and other intestinal pathogens using genome-wide RNAi libraries.

A Protocol to Infect Caenorhabditis elegans with Salmonella typhimurium

C. elegans has emerged as a new genetic model to study host-pathogen interactions. Here we describe a protocol to infect C. elegans with Salmonella typhimurium coupled with the double-strand RNAi interference technique to examine the role of host genes in defense against Salmonella infection.

In the last decade, C. elegans has emerged as an invertebrate organism to study interactions between hosts and pathogens, including the host defense ...

Neutrophil Isolation and Analysis to Determine their Role in Lymphoma Cell Sensitivity to Therapeutic Agents

Neutrophils are the most abundant (40% to 75%) type of white blood cells and among the first inflammatory cells to migrate towards the site of inflammation. They are key players in the innate immune system and play major roles in cancer biology. Neutrophils have been proposed as key mediators of malignant transformation, tumor progression, angiogenesis and in the modulation of the antitumor immunity; through their release of soluble factors or their interaction with tumor cells. To characterize the specific functions of neutrophils, a fast and reliable method is coveted for in vitro isolation of neutrophils from human blood. Here, a density gradient separation method is demonstrated to isolate neutrophils as well as mononuclear cells from the blood. The procedure consists of layering the density gradient solution such as Ficoll carefully above the diluted blood obtained from patients diagnosed with chronic lymphocytic leukemia (CLL), followed by centrifugation, isolation of mononuclear layer, separation of neutrophils from RBCsby dextran then lysis of residual erythrocytes. This method has been shown to isolate neutrophils &#8805; 90 % pure. To mimic the tumor microenvironment, 3-dimensional (3D) experiments were performed using basement membrane matrix such as Matrigel. Given the short half-life of neutrophils in vitro, 3D experiments with fresh human neutrophils cannot be performed. For this reason promyelocytic HL60 cells are differentiated along the granulocytic pathway using the differentiation inducers dimethyl sulfoxide (DMSO) and retinoic acid (RA). The aim of our experiments is to study the role of neutrophils on the sensitivity of lymphoma cells to anti-lymphoma agents. However these methods can be generalized to study the interactions of neutrophils or neutrophil-like cells with a large range of cell types in different situations.

Neutrophils are the most abundant type of white blood cells. The isolation of neutrophils from human blood by density gradient separation method and the differentiation of human promyelocytic (HL60) cells along the granulocytic pathway are described here; to test their role on sensitivity of lymphoma cells to anti-lymphoma agents.

Neutrophils are the most abundant (40% to 75%) type of white blood cells and among the first inflammatory cells to migrate towards the site of ...

Applying Fluorescence Resonance Energy Transfer (FRET) to Examine Effector Translocation Efficiency by Coxiella burnetii during siRNA Silencing

Coxiella burnetii, the causative agent of Q fever, is an intracellular pathogen that relies on a Type IV Dot/Icm Secretion System to establish a replicative niche. A cohort of effectors are translocated through this system into the host cell to manipulate host processes and allow the establishment of a unique lysosome-derived vacuole for replication. The method presented here involves the combination of two well-established techniques: specific gene silencing using siRNA and measurement of effector translocation using a FRET-based substrate that relies on &#946;-lactamase activity. Applying these two approaches, we can begin to understand the role of host factors in bacterial secretion system function and effector translocation. In this study we examined the role of Rab5A and Rab7A, both important regulators of the endocytic trafficking pathway. We demonstrate that silencing the expression of either protein results in a decrease in effector translocation efficiency. These methods can be easily modified to examine other intracellular and extracellular pathogens that also utilize secretion systems. In this way, a global picture of host factors involved in bacterial effector translocation may be revealed.

Applying Fluorescence Resonance Energy Transfer FRET to Examine Effector Translocation Efficiency by Coxiella burnetii during siRNA Silencing

Investigating the interactions between bacterial pathogens and their hosts is an important area of biological research. Here, we describe the necessary techniques to measure effector translocation by Coxiella burnetii during siRNA gene silencing using BlaM substrate.

Coxiella burnetii, the causative agent of Q fever, is an intracellular pathogen that relies on a Type IV Dot/Icm Secretion System to establish a ...

Mouse Models Of Helicobacter Infection And Gastric Pathologies

Helicobacter pylori is a gastric pathogen that is present in half of the global population and is a significant cause of morbidity and mortality in humans. Several mouse models of gastric Helicobacter infection have been developed to study the molecular and cellular mechanisms whereby H. pylori bacteria colonize the stomach of human hosts and cause disease. Herein, we describe protocols to: 1) prepare bacterial suspensions for the in vivo infection of mice via intragastric gavage; 2) determine bacterial colonization levels in mouse gastric tissues, by polymerase chain reaction (PCR) and viable counting; and 3) assess pathological changes, by histology. To establish Helicobacter infection in mice, specific pathogen-free (SPF) animals are first inoculated with suspensions (containing &#8805;105 colony-forming units, CFUs) of mouse-colonizing strains of either Helicobacter pylori or other gastric Helicobacter spp. from animals, such as Helicobacter felis. At the appropriate time-points post-infection, stomachs are excised and dissected sagittally into two equal tissue fragments, each comprising the antrum and body regions. One of these fragments is then used for either viable counting or DNA extraction, while the other is subjected to histological processing. Bacterial colonization and histopathological changes in the stomach may be assessed routinely in gastric tissue sections stained with Warthin-Starry, Giemsa or Haematoxylin and Eosin (H&#38;E) stains, as appropriate. Additional immunological analyses may also be undertaken by immunohistochemistry or immunofluorescence on mouse gastric tissue sections. The protocols described below are specifically designed to enable the assessment in mice of gastric pathologies resembling those in human-related H. pylori diseases, including inflammation, gland atrophy and lymphoid follicle formation. The inoculum preparation and intragastric gavage protocols may also be adapted to study the pathogenesis of other enteric human pathogens that colonize mice, such as Salmonella Typhimurium or Citrobacter rodentium.

Mouse Models Of Helicobacter Infection And Gastric Pathologies

Mice represent an invaluable in vivo model to study infection and diseases caused by gastrointestinal microorganisms. Here, we describe the methods used to study bacterial colonization and histopathological changes in mouse models of Helicobacter pylori-related disease.

Helicobacter pylori is a gastric pathogen that is present in half of the global population and is a significant cause of morbidity and mortality in ...

Evaluating Virulence and Pathogenesis of Aeromonas Infection in a Caenorhabditis elegans Model

The human pathogen Aeromonas has been clinically shown to cause gastroenteritis, wound infections, septicemia, and urinary tract infections. Most human diseases have been reported to be associated with four species of bacteria: Aeromonas dhakensis, Aeromonas hydrophila, Aeromonas veronii, and Aeromonas caviae. The model organism Caenorhabditis elegans is a bacterivore that provides an excellent infection model by which to study the bacterial pathogenesis of Aeromonas. Here, we introduce three different experiments to study Aeromonas infection using a C. elegans model, including survival, liquid toxicity, and muscle necrosis assays. The results of the three methods determining the virulence of Aeromonas were consistent. A. dhakensis was shown to be the most toxic among the 4 major Aeromonas species causing clinical infections. These methods are shown to be a convenient way to evaluate the toxicity among and within Aeromonas species and contribute to our understanding of the pathogenesis of Aeromonas infection.

Evaluating Virulence and Pathogenesis of Aeromonas Infection in a Caenorhabditis elegans Model

Here, we introduce three different experiments to study Aeromonas infection in C. elegans. Using these convenient methods, it is easy to evaluate the toxicity among and within Aeromonas species.

The human pathogen Aeromonas has been clinically shown to cause gastroenteritis, wound infections, septicemia, and urinary tract infections. Most human ...

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.

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.

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