Name: studying inherited immunity in a caenorhabditis elegans model of microsporidia infection
Uploaded: 2022-04-06T14:10:00
Description: Watch this Scientific Journal Video about Studying Inherited Immunity in a Caenorhabditis elegans Model of Microsporidia Infection at JoVE.com

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 OP5...

<ol>
	<li>Tetreau, G., Dhinaut, J., Gourbal, B., Moret, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Trans-generational+immune+priming+in+invertebrates:+current+knowledge+and+future+prospects.">Trans-generational immune priming in invertebrates: current knowledge and future prospects.</a> Frontiers in Immunology. 10, 1938 (2019).</li><li>Au, V., 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=CRISPR/Cas9+methodology+for+the+generation+of+knockout+deletions+in+Caenorhabditis+elegans.">CRISPR/Cas9 methodology for the generation of knockout deletions in Caenorhabditis elegans.</a> G3 Genes|Genomes|Genetics. 9 (1), 135-144 (2019).</li><li>Kamath, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genome-wide+RNAi+screening+in+Caenorhabditis+elegans.">Genome-wide RNAi screening in Caenorhabditis elegans.</a> Methods. 30 (4), 313-321 (2003).</li><li>The C. elegans Sequencing Consortium. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genome+sequence+of+the+nematode+C.+elegans:+a+platform+for+investigating+biology.">Genome sequence of the nematode C. elegans: a platform for investigating biology.</a> Science. 282 (5396), 2012-2018 (1998).</li><li>Yoshimura, 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=Recompleting+the+Caenorhabditis+elegans+genome.">Recompleting the Caenorhabditis elegans genome.</a> Genome Research. 29, 1009-1022 (2019).</li><li>Weinhouse, C., Truong, L., Meyer, J. N., Allard, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Caenorhabditis+elegans+as+an+emerging+model+system+in+environmental+epigenetics:+C.+elegans+as+an+environmental+epigenetics+model.">Caenorhabditis elegans as an emerging model system in environmental epigenetics: C. elegans as an environmental epigenetics model.</a> Environmental and Molecular Mutagenesis. 59 (7), 560-575 (2018).</li><li>Ermolaeva, M. A., Schumacher, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Insights+from+the+worm:+the+C.+elegans+model+for+innate+immunity.">Insights from the worm: the C. elegans model for innate immunity.</a> Seminars in Immunology. 26 (4), 303-309 (2014).</li><li>Willis, A. R., Sukhdeo, R., Reinke, A. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Remembering+your+enemies:+mechanisms+of+within-generation+and+multigenerational+immune+priming+in+Caenorhabditis+elegans.">Remembering your enemies: mechanisms of within-generation and multigenerational immune priming in Caenorhabditis elegans.</a> TheFEBS Journal. 288 (6), 1759-1770 (2020).</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=Cysteine+synthases+CYSL-1+and+CYSL-2+mediate+C.+elegans+heritable+adaptation+to+P.+vranovensis+infection.">Cysteine synthases CYSL-1 and CYSL-2 mediate C. elegans heritable adaptation to P. vranovensis infection.</a> Nature Communications. 11, 1741 (2020).</li><li>Wadi, L., Reinke, A. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evolution+of+microsporidia:+an+extremely+successful+group+of+eukaryotic+intracellular+parasites.">Evolution of microsporidia: an extremely successful group of eukaryotic intracellular parasites.</a> PLoS Pathogens. 16, 1008276 (2020).</li><li>Han, B., Takvorian, P. M., Weiss, L. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Invasion+of+host+cells+by+microsporidia.">Invasion of host cells by microsporidia.</a> Frontiers in Microbiology. 11, 172 (2020).</li><li>Tamim El Jarkass, H., Reinke, A. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+ins+and+outs+of+host-microsporidia+interactions+during+invasion,+proliferation+and+exit.">The ins and outs of host-microsporidia interactions during invasion, proliferation and exit.</a> Cellular Microbiology. 22 (11), 13247 (2020).</li><li>Balla, K. M., La&#382;eti&#263;, V., 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=Natural+variation+in+the+roles+of+C.+elegans+autophagy+components+during+microsporidia+infection.">Natural variation in the roles of C. elegans autophagy components during microsporidia infection.</a> PLoS ONE. 14, 0216011 (2019).</li><li>Szumowski, S. C., Estes, K. A., 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=Preparing+a+discreet+escape:+Microsporidia+reorganize+host+cytoskeleton+prior+to+non-lytic+exit+from+C.+elegans+intestinal+cells.">Preparing a discreet escape: Microsporidia reorganize host cytoskeleton prior to non-lytic exit from C. elegans intestinal cells.</a> Worm. 1 (4), 207-211 (2012).</li><li>Balla, K. M., Luallen, R. J., Bakowski, M. A., 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=Cell-to-cell+spread+of+microsporidia+causes+Caenorhabditis+elegans+organs+to+form+syncytia.">Cell-to-cell spread of microsporidia causes Caenorhabditis elegans organs to form syncytia.</a> Nature Microbiology. 1 (11), 1-6 (2016).</li><li>Reinke, A. W., Balla, K. M., Bennett, E. J., 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=Identification+of+microsporidia+host-exposed+proteins+reveals+a+repertoire+of+rapidly+evolving+proteins.">Identification of microsporidia host-exposed proteins reveals a repertoire of rapidly evolving proteins.</a> Nature Communications. 8, 14023 (2017).</li><li>Bakowski, M. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ubiquitin-mediated+response+to+microsporidia+and+virus+infection+in+C.+elegans.">Ubiquitin-mediated response to microsporidia and virus infection in C. elegans.</a> PLoS Pathogen. 10, 1004200 (2014).</li><li>Reddy, K. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+intracellular+pathogen+response+pathway+promotes+proteostasis+in+C.+elegans.">An intracellular pathogen response pathway promotes proteostasis in C. elegans.</a> Current Biology. 27 (22), 3544-3553 (2017).</li><li>Willis, A. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+parental+transcriptional+response+to+microsporidia+infection+induces+inherited+immunity+in+offspring.">A parental transcriptional response to microsporidia infection induces inherited immunity in offspring.</a> Science Advances. 7 (19), (2021).</li><li>Tamim El Jarkass, H., 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=An+intestinally+secreted+host+factor+promotes+microsporidia+invasion+of+C.+elegans.">An intestinally secreted host factor promotes microsporidia invasion of C. elegans.</a> eLife. 11, 72458 (2022).</li><li>Solis, G. M., Petrascheck, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measuring+Caenorhabditis+elegans+life+span+in+96+well+microtiter+plates.">Measuring Caenorhabditis elegans life span in 96 well microtiter plates.</a> Journal of Visualized Experiments. 49, 2496 (2011).</li><li>Stiernagle, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Maintenance+of+C.+elegans.">Maintenance of C. elegans.</a> WormBook. , (2006).</li><li>Sutphin, G. L., Kaeberlein, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measuring+Caenorhabditis+elegans+life+span+on+solid+media.">Measuring Caenorhabditis elegans life span on solid media.</a> Journal of Visualized Experiments. (27), e1152 (2009).</li><li>Estes, K. A., Szumowski, S. 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. 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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 ...

These protocols enable the study of inherited immunity to microsporidia. Techniques explained here can help us understand how immunity is transmitted across generations and the molecules determining immunity to microsporidia in offspring. C.elegans is a simple and genetically tractable model with a short generation time for assaying inheritance.The many tools available for the worm can be used to uncover molecular mechanisms of intergenerational immunity. When infecting P0 animals, use a low spore dose that does not significantly impact the parent's ability to produce progeny. This ensures that bleaching yields enough F1 animals for downstream experiments To begin, move the required number of 10-centimeter unseeded nematode growth medium plates from four degrees Celsius to room temperature one day before planned infections.Just before the infection, pellet down approximately 2, 500 synchronized L1 worms in a microfuge tube at 1, 400 times G for 30 seconds. Remove the supernatant with a pipette tip to leave worms in 50 microliters of M9 media. Add one milliliter of 10X Escherichia coli strain OP50 to the 1-stage worms, and N.parisii spores to the desired concentration.As an uninfected control, prepare an equivalent tube of L1s and OP50 with a volume of M9 media equivalent to the volume of spore preparation used. Mix the L1 worm sample by vortexing briefly and plate above one milliliter of worms onto a 10-centimeter unseeded nematode growth medium plate. Swirl to ensure the liquid spreads over the whole plate.Dry the plates in a clean cabinet with the lids off for 10 to 20 minutes or until completely dry before incubating at 21 degrees Celsius for 72 hours. At 72 hours post-infection, examine plates under the dissecting microscope. The plate with infected worms should contain fewer embryos than the plates with uninfected worms.Then wash the worms off the plates into a microcentrifuge tube using one milliliter M9 media. If many worms remain on the plates, pellet the worms by centrifugation at 1400 times G for 30 seconds. Remove the supernatant and perform additional plate washes.Centrifuge at 1400 times G for 30 seconds at room temperature to pellet the worms and wash twice with one milliliter of M9 media or until the supernatant is clear. Re-suspend it in a final volume of one milliliter M9 media and mix well by pipetting. Transfer 100 microliters of the suspended worms to a fresh tube and bleach the remaining 900 microliters to break open the adult worms and release F1 embryos for testing.Perform infections with modifications. as mentioned in the manuscript. At 72 hours post-infection, examine plates under a dissecting microscope.The plate with naive worms should be smaller and contain less embryos than the plates with primed worms. Then, start the staining and fixing process of worms by washing them off the plates into a microcentrifuge tube using one milliliter of 0.1%Tween 20 in M9 media. If many worms remain on the plates, pellet the worms by centrifugation at 1, 400 times G for 30 seconds at room temperature, remove the supernatant using a pipette tip, and perform additional plate washes.After pelleting the worms by centrifuging for 30 seconds at 1, 400 times G at room temperature, wash twice with one milliliters of M9 media containing 0.1%Tween 20 or until the supernatant is clear. Remove the supernatant, add 700 microliters of acetone, and leave the worms to fix at room temperature for 10 minutes. Pellet out these fixed worms for 30 seconds at 10, 000 times G at room temperature and give two washes of one milliliter phosphate buffer saline containing 0.1%Tween 20.Prepare 50 milliliters of DY96 working solution, wrap the container in foil, and store it in a drawer to prevent exposure to light. Add 500 microliters of DY96 working solution to the worm pellet and rotate for 30 minutes at room temperature in darkness. Centrifuge this reaction for 30 seconds at 10, 000 times G at room temperature to pellet the worms and remove the supernatant.Then, add 15 microliters of the mounting medium with or without DAPI. Pipette 10 microliters of these stained worms onto a microscope slide and place a cover slip over the top. To analyze the DY96-stained worms mounted on slides, perform imaging using the GFP channel of a fluorescence microscope.To determine the fitness of the worm populations, use a 5X or 10X objective to assay the gravidity of more than 100 worms per condition. At 72 hours post-infection, the F1 animals were fixed and stained with DY96 to assess microsporidia resistance. Infected parental populations and uninfected controls were fixed at 72 hours post-infection and stained with DY96 to visualize the worm embryos and microsporidia spores.Infected animals are small, contain many microsporidia spores, and produce fewer embryos than healthy, uninfected controls. Assessment of worm gravidity showed that approximately 95%of uninfected animals produced offspring, compared to the infected animals, which were less than 80%Quantifications revealed that approximately 90%of the microsporidia-treated population were infected, as determined by the number of worms containing DY96-stained spores. Quantifications of these fixed animals revealed that the primed worms contained significantly more embryos than their naive counterparts, indicating greater fitness in the face of infection.Fiji ImageJ was used to determine the parasite burden of individual naive and immune primed worms, which is the percentage of the body filled with fluorescent N.parisii spores. Quantifications revealed a dramatic reduction in the parasite burden of worms that came from infected parents. Further, the calculations of individual worm size revealed that primed worms had a significant growth advantage over naive animals in the face of N.parisii infection.Imaging studies revealed that while naive animals typically contained multiple spores and several infected cells, the primed animals had far fewer or no spores and typically no sporoplasms. If unprimed and primed F1 populations have similar numbers of gravid and infected animals, count the number of eggs per animal, and analyze infected tissue with ImageJ to find more subtle differences. While direct yellow 96 stains mature spores, fluorescence in situ hybridization can reveal sporoplasms within cells.This can show you the number of infected cells in primed and unprimed worms. These techniques have shown that the parental transcriptional response can be instrumental in inducing inherited immunity in offspring. Researchers are now investigating the nature of this immunity in offspring.

Research

JoVE Journal

Immunology and Infection

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