We are grateful for the assistance from the C. elegans core facility in Taiwan and to the Diagnostic Microbiology and Antimicrobial Resistance Laboratory of National Cheng Kung University Hospital for providing the Aeromonas isolates. We also acknowledge the Caenorhabditis Genetics Center (CGC), and the WormBase. We also thank Savana Moore for editing the manuscript.
This study was partially supported by grants from the Ministry of Science and Technology of Taiwan (MOST 105-2628-B-006-017-MY3) and the National Cheng Kung University Hospital (NCKUH-10705001) to P.L. Chen.

1. Preparation of the Culture Medium
NOTE: See Table 1 for solution preparation.
<ol>
	<li>To prepare M9 medium12, dissolve 1.5 g of KH2PO4, 5.66 g of Na2HPO4, and 2.5 g of NaCl in 500 mL of deionized water. Autoclave at 121 &#176;C for 20 min. Wait until cooled to room temperature and then add 0.5 mL of 1 M MgSO4 before first use.</li>
	<li>To prepare nematode growth medium (NGM)12, dissolve 3 g NaCl, 2.5 g bacterial peptone, and 20 g agar in 1 L of deionized water. Autoclave at 121 &#176;C for 20 min. Wait until cooled to 55 &#176;C in a water bath and then add 1 mL of 1 M CaCl2, 1 mL of 1 M MgSO4, 1 mL of 5% cholesterol in ethanol, and 25 mL of phosphate buffer. Swirl to mix well.</li>
	<li>Pour about 5 mL NGM into each 6 cm Petri dish. Avoid bubble production on the surface of plate. Leave plates at room temperature for 1 night and package to save at 4 &#176;C. Bring back to room temperature before use.</li>
	<li>To prepare enriched NGM (ENGM), dissolve 3 g NaCl, 5 g bacterial peptone, 1 g yeast extract, and 30 g agar in 1 L of deionized water. Autoclave at 121 &#176;C for 20 min. Wait until cooled to 55 &#176;C in a water bath, and add 1 mL of 1 M CaCl2, 1 mL of 1 M MgSO4, 1 mL of 5% cholesterol in ethanol, and 25 mL of phosphate buffer. Swirl to mix well.</li>
	<li>Pour about 10 mL ENGM into each 9 cm Petri dish. Avoid bubble production on the surface of plate. Leave plates at room temperature for 1 night and package to save at 4 &#176;C. Bring to room temperature before use.</li>
	<li>To prepare S medium12, mix 40 mL S Basal, 0.4 mL 1 M potassium citrate, 0.4 mL trace metals solution, 0.12 mL of 1 M CaCl2, 0.12 mL of 1 M MgSO4, 40 &#181;L of 5% cholesterol in ethanol and 1 mL of 8 mM FudR before use.</li>
</ol>
2. Synchronization of C. elegans6,9,12
<ol>
	<li>Wash about 2,000 worms in the gravid-adult stage with 10 mL of sterile deionized water in a 15 mL tube. Wash out the bacteria 3x, keeping the worms at the bottom of the tube.</li>
	<li>Centrifuge the sample for 1 min at 500 x g to pull down the worms. Remove the supernatant, and keep the worms in 3.5 mL deionized water.</li>
	<li>Add 1 mL NaOCl (10&#8211;15%) and 0.5 mL 5 M KOH into the tube. Mix evenly by shaking for &#60;6 min to lyse the worm bodies.</li>
	<li>After the eggs are released from the worms, add 10 mL deionized water to stop the lysis. Centrifuge for 1 min at 1,200 x g to pull down the eggs and remove the supernatant as much as possible. Wash with 15 mL M9 medium at least 3x. 
	NOTE: See step 1.1 for the composition of M9 medium.</li>
	<li>Keep eggs in 1 mL M9 medium. Transport eggs to a 3.5 cm dish for incubation at 20 &#176;C for one night (about 12&#8211;18 h). 
	NOTE: After the incubation, the eggs will hatch to worm larva which is called as first larva (L1) stage.</li>
	<li>Pipette out 10 &#181;L of L1 worms in M9 medium. Count the worm number and calculate the concentration of L1 worms in M9 medium.</li>
	<li>Seed at most 10,000 incubated L1 stage worms with a pipet on a 9 cm ENGM plate spread with Escherichia coli OP50.
	<ol>
		<li>In this step, spread 0.5 mL overnight cultured E. coli OP50 with Luria-Bertani (LB) broth on the 9 cm ENGM plate. Incubate the plate at 37 &#176;C for 16&#8211;18 h 
		NOTE: To avoid contamination, the spreading step should be performed in a laminar flow hood.</li>
		<li>Cool to room temperature before seeding L1 worms. Seed at most 10,000 incubated L1 stage worms on the&#160;ENGM plate with E. coli OP50.</li>
	</ol>
	</li>
	<li>Incubate the worms at 20 &#176;C for 44 h or until they grow to the 4th larva (L4) stage. 
	NOTE: See step 1.3 for the composition of ENGM medium. A worm has a white dot in half-moon shape at the middle of body side at the L4 stage.</li>
	<li>Use the synchronized L4 stage worms in the following assays. Use wild type N2 worms in the C. elegans survival assay with Aeromonas dhakensis and the C. elegans liquid toxicity assay with Aeromonas dhakensis. Use RW1596 worms (myo-3(st386);stEx30[myo-3p::GFP::myo-3 + rol-6(su1006)]) in the C. elegans muscle necrosis assay with Aeromonas dhakensis.</li>
</ol>
3. C. elegans Survival Assay with Aeromonas6,9
<ol>
	<li>On the day of seeding L1 worms on the ENGM spreading with E. coli OP50, pick a single colony of each of the four Aeromonas strains or E. coli OP50 and culture with 2 mL LB broth respectively at 37 &#176;C for 16 h. 
	NOTE: To avoid contamination, this step should be performed in a laminar flow hood. Here, the following bacterial strains were used: E. coli OP50, the normal food source of C. elegans. A. dhakensis AAK1, the first fully-sequenced pathogenic clinical&#160;A. dhakensis isolate. A. hydrophila A2-066, A. veronii A2-007, and A. caviae A2-9307121 are representatively clinical isolates from National Cheng Kung University Hospital.</li>
	<li>Measure the OD600 absorbance of bacterial broth and adjust the bacterial broth to OD600 = 2.0 with LB broth.</li>
	<li>Spot and spread 30 &#181;L of bacterial broth each 6 cm NGM plate. Culture the plates at room temperature overnight. 
	NOTE: See step 1.2 for the composition of NGM medium.</li>
	<li>On the day the L1 worms grow to the L4 stage, randomly pick and transfer 50 worms to an NGM plate with each of the four Aeromonas strains or E. coli OP50.</li>
	<li>Incubate the NGM plates at 20 &#176;C until the assay is finished.</li>
	<li>Transfer all the living worms to a newly prepared NGM plate with bacteria and count the numbers of live, dead, and sensor worms every day until the last worm is dead. 
	NOTE: Transfer worms to a fresh-prepared NGM plate every day for the maintenance of infection, even if using sterile worms (e.g., glp-4, or with FudR).</li>
	<li>Plot the survival curves based on the daily data calculation.</li>
</ol>
4. C. elegans Liquid Toxicity Assay with Aeromonas7
NOTE: To avoid contamination, steps should be performed in a laminar flow hood.
<ol>
	<li>The day after seeding the L1 worms on the ENGM plate spread with E. coli OP50, pick a single colony of each of the four Aeromonas strains and E. coli OP50, and culture with 5 mL LB broth each at 37 &#176;C for 16 h. 
	NOTE: In the protocol, the following bacteria strains were used: E. coli OP50, the normal food source of C. elegans. A. dhakensis AAK1, the first fully-sequenced pathogenic clinical A. dhakensis isolate. A. hydrophila A2-066, A. veronii A2-007, and A. caviae A2-9307121 are representatively clinical isolates from National Cheng Kung University Hospital.</li>
	<li>Measure the OD600 absorbance of the bacteria broth.</li>
	<li>Centrifuge the bacterial broth at 3,500 x g for 15 min to remove the LB broth. Resuspend the bacteria and adjust the bacterial broth to OD600 = 3.0 with S medium.Add 195 &#181;L of bacterial broth in S medium to at least 8 wells of a 96-well plate. 
	NOTE: See step 1.6 for the composition of S medium.</li>
	<li>Wash off the L4 worms on the ENGM plate with E. coli OP5 using M9 medium. Adjust the worm solution to a concentration of 5 worms per &#181;L and add 5 &#181;L of the worm solution to each well of the 96-well plate. Ensure that there are approximately 25 worms per well.</li>
	<li>Incubate the 96-well plate on a shaker at 200 rpm at 25 &#176;C.</li>
	<li>Count the number of live worms and dead worms after 24, 48, and 72 h. Calculate the survival rates of each well with the following formula: (Live worms/Total worms) &#215; 100%.</li>
	<li>Draw a scatter plot graph with the daily data calculation.</li>
</ol>
5. C. elegans Muscle Necrosis Assay with Aeromonas6
NOTE: To avoid contamination, these steps should be performed in a laminar flow hood.
<ol>
	<li>On the day the L1 worms are seeded on the ENGM spread with E. coli OP50, pick a single colony of each of the four Aeromonas strains and E. coli OP50 and culture with 2 mL LB broth each at 37 &#176;C for 16 h. 
	&#8203;NOTE: Here, the following bacteria strains were used: E. coli OP50, the normal food source of C. elegans. A. dhakensis AAK1, the first fully-sequenced pathogenic clinical&#160;A. dhakensis isolate. A. hydrophila A2-066, A. veronii A2-007, and A. caviae A2-9307121 are representatively clinical isolates from National Cheng Kung University Hospital.</li>
	<li>Measure the OD600 absorbance of the bacteria broth and adjust the bacteria broth to OD600 = 2.0 with LB broth. Spot and spread 30 &#181;L of bacterial broth on 6 cm NGM plates. Culture the plates at room temperature overnight.</li>
	<li>On the day the L1 worms grow to the L4 stage, transfer 50 worms to each NGM plate with each of the four Aeromonas strains or E. coli OP50. Incubate the NGM plates at 20 &#176;C.Transfer worms every 24 h to newly prepared NGM plates with bacteria.</li>
	<li>Take muscle images using a fluorescence microscope.
	<ol>
		<li>Randomly pick 10 worms onto a 2% agarose gel in M9 medium on a slide. Paralyze the worms using 2 &#181;L of 1% sodium azide in M9 medium on agarose for less than 5 min before taking images.</li>
		<li>Place a cover slip and capture the muscle images using a green fluorescent protein (GFP) filter on a fluorescent microscope with charge-coupled device camera. Capture the muscle images at 24, 48, and 72 h post the L4 stage.</li>
	</ol>
	</li>
	<li>Conduct image analysis and figure preparation with an image processing software. 
	NOTE: The definitions of the scores and the muscle damage levels are shown in Figure 3, for which the criteria are listed as follows: 3 points for missing muscle fibers; 2 points for ruptured or broken muscle fibers; 1 point for bent muscle fibers; 0 points for healthy muscle fibers.</li>
</ol>
6. Statistical Analyses
<ol>
	<li>Perform all experiments a minimum of three times independently.</li>
	<li>Use the Kaplan-Meier method to assess the C. elegans survival assays, and use the log-rank test in analyzing survival differences. Set statistical significance at 0.05.</li>
	<li>Use Student&#39;s t-test to analyze the statistical results of the C. elegans liquid toxicity assays for the two different groups. Use one-way ANOVA test in analyzing differences among three or more values for one independent variable. Set statistical significance at 0.05.</li>
</ol>

<ol>
	<li>Parker, J. L., Shaw, J. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Aeromonas+spp.+clinical+microbiology+and+disease.">Aeromonas spp. clinical microbiology and disease.</a> Journal of Infection. 62 (2), 109-118 (2011).</li><li>Chao, C. M., Lai, C. C., Tang, H. J., Ko, W. C., Hsueh, P. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Skin+and+soft-tissue+infections+caused+by+Aeromonas+species.">Skin and soft-tissue infections caused by Aeromonas species.</a> European Journal of Clinical Microbiology &amp; Infectious Diseases. 32 (4), 543-547 (2013).</li><li>Chao, C. M., Lai, C. C., Tang, H. J., Ko, W. C., Hsueh, P. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biliary+tract+infections+caused+by+Aeromonas+species.">Biliary tract infections caused by Aeromonas species.</a> European Journal of Clinical Microbiology &amp; Infectious Diseases. 32 (2), 245-251 (2013).</li><li>Chuang, H. 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=Different+clinical+characteristics+among+Aeromonas+hydrophila,+Aeromonas+veronii+biovar+sobria+and+Aeromonas+caviae+monomicrobial+bacteremia.">Different clinical characteristics among Aeromonas hydrophila, Aeromonas veronii biovar sobria and Aeromonas caviae monomicrobial bacteremia.</a> Journal of Korean Medical Science. 26 (11), 1415-1420 (2011).</li><li>Chao, C. M., Lai, C. C., Gau, S. J., Hsueh, P. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Skin+and+soft+tissue+infection+caused+by+Aeromonas+species+in+cancer+patients.">Skin and soft tissue infection caused by Aeromonas species in cancer patients.</a> Journal of Microbiology, Immunology and Infection. 46 (2), 144-146 (2013).</li><li>Chen, P. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Disease+Model+of+Muscle+necrosis+caused+by+Aeromonas+dhakensis+infection+in+Caenorhabditis+elegans.">A Disease Model of Muscle necrosis caused by Aeromonas dhakensis infection in Caenorhabditis elegans.</a> Frontiers in Microbiology. 7, 2058 (2016).</li><li>Chen, P. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Virulence+diversity+among+bacteremic+Aeromonas+isolates:+ex+vivo,+animal,+and+clinical+evidences.">Virulence diversity among bacteremic Aeromonas isolates: ex vivo, animal, and clinical evidences.</a> PLoS One. 9 (11), 111213 (2014).</li><li>Saraceni, P. R., Romero, A., Figueras, A., Novoa, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Establishment+of+infection+models+in+zebrafish+larvae+(Danio+rerio)+to+study+the+pathogenesis+of+Aeromonas+hydrophila.">Establishment of infection models in zebrafish larvae (Danio rerio) to study the pathogenesis of Aeromonas hydrophila.</a> Frontiers in Microbiology. 7, 1219 (2016).</li><li>Chen, Y. W., Ko, W. C., Chen, C. S., Chen, P. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RIOK-1+Is+a+Suppressor+of+the+p38+MAPK+Innate+Immune+Pathway+in+Caenorhabditis+elegans.">RIOK-1 Is a Suppressor of the p38 MAPK Innate Immune Pathway in Caenorhabditis elegans.</a> Frontiers in Immunology. 9, 774 (2018).</li><li>Powell, J. R., 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=Models+of+Caenorhabditis+elegans+infection+by+bacterial+and+fungal+pathogens.">Models of Caenorhabditis elegans infection by bacterial and fungal pathogens.</a> Methods in Molecular Biology. 415, 403-427 (2008).</li><li>Chou, T. 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=Enterohaemorrhagic+Escherichia+coli+O157:H7+Shiga-like+toxin+1+is+required+for+full+pathogenicity+and+activation+of+the+p38+mitogen-activated+protein+kinase+pathway+in+Caenorhabditis+elegans.">Enterohaemorrhagic Escherichia coli O157:H7 Shiga-like toxin 1 is required for full pathogenicity and activation of the p38 mitogen-activated protein kinase pathway in Caenorhabditis elegans.</a> Cellular Microbiology. 15 (1), 82-97 (2013).</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. , 1-11 (2006).</li><li>Engelmann, I., Pujol, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Innate+immunity+in+C.+elegans.">Innate immunity in C. elegans.</a> Advances in Experimental Medicine and Biology. 708, 105-121 (2010).</li><li>Feinbaum, R. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genome-wide+identification+of+Pseudomonas+aeruginosa+virulence-related+genes+using+a+Caenorhabditis+elegans+infection+model.">Genome-wide identification of Pseudomonas aeruginosa virulence-related genes using a Caenorhabditis elegans infection model.</a> PLoS Pathogens. 8 (7), 1002813 (2012).</li></ol>

The authors have nothing to disclose.

C. elegans is a bacterivorous nematode that naturally intakes bacteria as food and has developed a complicated innate immunity to bacteria during its evolutionary process. Two of the major organs maintaining and supporting the immunity are the epidermis and intestine9,13. The epidermis and bands of muscle of C. elegans resemble the soft-tissue structures in mammals and humans6. Because of these characteristics, C. elegans is applicable as a model organism for studying the pathogenesis of Aeromonas infection.
Here, we present three methods for examining Aeromonas infection in C. elegans that are representative of the diverse toxicity among 4 Aeromonas species and are correlated with clinical observation7. These convenient methods distinguish the different degrees of toxicity among the Aeromonas species. Using these methods, researchers can study the virulence of different Aeromonas species and the defending mechanism of hosts upon Aeromonas infection.
In these three methods, C. elegans acts not only as a predator but also as a prey of Aeromonas. If the worms are not in healthy condition before test, the results of host-pathogen interaction will be different. Thus, it is important to be very careful when manipulating C. elegans. To avoid excessive damage to the eggs of C. elegans in the synchronizing step, be sure to observe the release of eggs continuously. Once the eggs are damaged, the hatching number of C. elegans eggs will largely decrease. Even if the damaged eggs hatch successfully, the worms are likely to have developmental problems. Therefore, it is critical to complete step 2.3 within 6 min after the worms are exposed to NaOCl and KOH.
The three methods described above show cohesive results on the Aeromonas toxicities to C. elegans, including two different experiments measuring the survival rates of C. elegans under Aeromonas infection and one observing the morphological change of muscle. The virulence test using different designed methods may provide diverse results. For example, scientists have previously observed different outcomes in C. elegans plate and liquid assays14. In our research, we also found there was discrepancy existing between these two assays9. It is believed that the external environment will influence virulence of the pathogen, and the researchers need to find the optimal conditions to explore the virulence mechanism. From another perspective, it is worth studying and understanding the underlying mechanisms in specific condition of infection.
In this work, we established an infection model in C. elegans to study the virulence of Aeromonas and the corresponding host response after infection. These three methods could be applied to study the other Aeromonas species beyond the 4 species addressed in the present study. In addition, these approaches will provide a platform to study the underlying mechanism responsible for muscle necrosis induced by bacterial infection and to develop potential therapies to improve the clinical outcomes of severe soft tissue infections.

The human pathogen, Aeromonas, has been shown clinically to cause gastroenteritis, wound infections, septicemia, and urinary tract infections1,2. Most associated human diseases have been reported to be associated with four bacterial species: Aeromonas dhakensis, Aeromonas hydrophila, Aeromonas veronii, and Aeromonas caviae 2,3,4,5. Among Aeromonas infectious diseases, soft-tissue infections can cause severe morbidity and mortality in humans. Of note, muscle necrosis is the most severe form of soft tissue infection6. Observation of the survival and muscle necrosis of Caenorhabditis elegans after infection is a convenient method by which to speculate the toxicity of Aeromonas.
Scientists have already developed numerous model organisms to study bacterial infections. In previous studies, mice, zebrafishes, and nematodes were used as animal models to study the pathogenesis and virulence of Aeromonas6,7,8. Every animal model has its&#39; advantages and applications. The model organism, Caenorhabditis elegans, is a bacterivorous nematode which intakes bacteria as foodnaturally.&#160;C. eleganshas developed a complicated innate immune system against bacterial infection over the course of its evolution. Under the stress of bacterial infection,&#160;C. elegans has been proven to be an excellent infection model to study the bacterial pathogenesis of Aeromonas6,7,9 and other pathogens like fungus10 and enterohaemorrhagic Escherichia coli O157:H711. However, there is still no publication that focuses on the methodology in using C. elegans as a model for studying the virulence of Aeromonas.
Here, we introduce three different experiments to study Aeromonas infection using C. elegans as an animal model: assays for survival, liquid toxicity, and muscle necrosis. These methods are a convenient way to evaluate the toxicity among and within Aeromonas species and improve the understanding of the pathogenesis of Aeromonas.

<table><tbody><tr><td>Shaker incubator</td><td>YIH DER</td><td>LM-570R</td><td>Bacteria incubation</td></tr><tr><td>K&lt;sub&gt;2&lt;/sub&gt;HPO&lt;sub&gt;4&lt;/sub&gt;</td><td>J.T.Baker</td><td>MP021519455</td><td>Culture medium preparation&amp;nbsp;</td></tr><tr><td>KH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;</td><td>J.T.Baker</td><td>3246-05</td><td>Culture medium preparation&amp;nbsp;</td></tr><tr><td>Na&lt;sub&gt;2&lt;/sub&gt;HPO&lt;sub&gt;4&lt;/sub&gt;</td><td>J.T.Baker</td><td>MP021914405</td><td>Culture medium preparation&amp;nbsp;</td></tr><tr><td>NaCl</td><td>SIGMA</td><td>31434</td><td>Culture medium preparation&amp;nbsp;</td></tr><tr><td>MgSO&lt;sub&gt;4&lt;/sub&gt;</td><td>SIGMA</td><td>M7506</td><td>Culture medium preparation&amp;nbsp;</td></tr><tr><td>agar</td><td>Difco</td><td>214530</td><td>Culture medium preparation&amp;nbsp;</td></tr><tr><td>CaCl&lt;sub&gt;2&lt;/sub&gt;</td><td>SIGMA</td><td>C1016</td><td>Culture medium preparation&amp;nbsp;</td></tr><tr><td>cholesterol</td><td>SIGMA</td><td>C8503</td><td>Culture medium preparation&amp;nbsp;</td></tr><tr><td>ethanol</td><td>SIGMA</td><td>32205</td><td>Culture medium preparation&amp;nbsp;</td></tr><tr><td>KOH</td><td>SIGMA</td><td>P5958</td><td>Culture medium preparation&amp;nbsp;</td></tr><tr><td>6 cm Petri plate</td><td>ALPHA PLUS</td><td>46</td><td>agar plate preparation</td></tr><tr><td>96-well plate</td><td>FALCON</td><td>353072</td><td>liquid assay</td></tr><tr><td>bacterial peptone</td><td>Affymetrix/USB</td><td>AAJ20048P2</td><td>Culture medium preparation&amp;nbsp;</td></tr><tr><td>yeast extract</td><td>SIGMA</td><td>92144</td><td>Culture medium preparation&amp;nbsp;</td></tr><tr><td>citric acid&amp;bull;H&lt;sub&gt;2&lt;/sub&gt;O</td><td>SIGMA</td><td>C1909</td><td>Culture medium preparation&amp;nbsp;</td></tr><tr><td>tri-potassium citrate&amp;bull;H&lt;sub&gt;2&lt;/sub&gt;O</td><td>SIGMA</td><td>104956</td><td>Culture medium preparation&amp;nbsp;</td></tr><tr><td>FudR&amp;nbsp;</td><td>SIGMA</td><td>1271008</td><td>Culture medium preparation&amp;nbsp;</td></tr><tr><td>disodium EDTA</td><td>SIGMA</td><td>E1644</td><td>Culture medium preparation&amp;nbsp;</td></tr><tr><td>FeSO&lt;sub&gt;4&lt;/sub&gt;&amp;bull;7 H&lt;sub&gt;2&lt;/sub&gt;O</td><td>SIGMA</td><td>215422</td><td>Culture medium preparation&amp;nbsp;</td></tr><tr><td>MnCl&lt;sub&gt;2&lt;/sub&gt;&amp;bull;4 H&lt;sub&gt;2&lt;/sub&gt;O</td><td>SIGMA</td><td>221279</td><td>Culture medium preparation&amp;nbsp;</td></tr><tr><td>ZnSO&lt;sub&gt;4&lt;/sub&gt;&amp;bull;7 H&lt;sub&gt;2&lt;/sub&gt;O</td><td>SIGMA</td><td>204986</td><td>Culture medium preparation&amp;nbsp;</td></tr><tr><td>CuSO&lt;sub&gt;4&lt;/sub&gt;&amp;bull;5 H&lt;sub&gt;2&lt;/sub&gt;O</td><td>SIGMA</td><td>C8027</td><td>Culture medium preparation&amp;nbsp;</td></tr><tr><td>tryptone</td><td>SIGMA</td><td>16922</td><td>Culture medium preparation&amp;nbsp;</td></tr><tr><td>Microscope system</td><td>Nikon&amp;nbsp;</td><td>Eclipase Ti inverted&amp;nbsp;</td><td>microscope imaging</td></tr><tr><td>Scientific CCD Camera</td><td>QImaging&amp;nbsp;</td><td>Retiga-2000R Fast 1394&amp;nbsp;</td><td>microscope imaging</td></tr></tbody></table>

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.

By following the protocols described above, it is easy to differentiate between the toxicities from the four Aeromonas strains. The survival assay of C. elegans is shown in Figure 1. The survival rates of C. elegans infected with Aeromonas species, shown in order from high to low were: A. caviae, A. veronii, A. hydrophila, and A. dhakensis. Although there is diversity in terms of toxicity among and within Aeromonas species, the survival rates of C. elegans, on average, showed highest mortality among the worms infected with A. dhakensis. The virulence of A. hydrophila was inferior to that of A. dhakensis (p &#60; 0.01). In contrast to A. dhakensis and A. hydrophila,the attenuated virulences of both A. veronii and A. caviae were observed in the C. elegans survival assay. The diversity of the survival rates of C. elegans infected with different Aeromonas species is consistent with clinical observations of Aeromonas infection7.
The results of the liquid toxicity assay of C. elegans are shown in Figure 2. With the infection time, the numbers of worms that survived decreased significantly when C. elegans was infected with either A. dhakensis or A. hydrophila. The survival rate of the worms infected with A. dhakensis was inferior to those infected with A. hydrophila. In contrast, the groups infected with A. veronii or A. caviae had higher survival rates at each time point as compared to A. dhakensis and A. hydrophila. The trends of survival rates of C. elegans in the liquid toxicity assay representing the virulence of different Aeromonas species are similar to those observed in the survival assay.
In the C. elegans muscle necrosis assay, we first classified the levels of muscle damage and assigned scores according to the corresponding levels. The definitions of the score and the muscle damage levels are provided in Figure 3. The scoring criteria for each muscle damage level is as follows: 0 points for healthy muscle fibers; 1 point for bent muscle fibers; 2 points for ruptured or broken muscle fibers; 3 points for loss of muscle fibers. The results of the C. elegans muscle necrosis assay in Aeromonas infected-C. elegans is shown in Figure 4. The degrees of muscle damage varied among the Aeromonas species. The levels of muscle damage ranged in order from mild to severe as follows: A. caviae, A. veronii, A. hydrophila, and A. dhakensis at each time point. In contrast with A. hydrophila and A. dhakensis, muscle necrosis was not obvious in worms infected with A. caviae and A. veronii. Most of the worms infected with A. caviae and A. veronii were recorded as 0 points. C. elegans infected with A. hydrophila or A. dhakensis exhibited severe muscle damage. Of note, most of the worms infected with A. dhakensis demonstrated muscle damage graded &#8805;2 points. The degree of muscle necrosis of C. elegans infected with A. dhakensis was more severe in terms of a higher percentage of scores &#8805; 2 points compared with A. hydrophila. The severity of muscle necrosis in the 4 Aeromonas species correlated with the findings from the survival and liquid toxicity assays.
The methods described above offer a convenient way to differentiate virulence diversity among and within Aeromonas species. In addition, this is a reliable model by which to study the interaction between a pathogen and host.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/58768/58768fig1.jpg" /> 
Figure 1: The survival curves of C. elegans. The survival assay shows the diverse toxicities of different Aeromonas species to C. elegans (**: P &#60; 0.01; *: P &#60; 0.05). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58768/58768fig1large.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/58768/58768fig2.jpg" /> 
Figure 2: The survival rate of C. elegans from the liquid toxicity assay. The liquid toxicity assay shows the different toxicities of various Aeromonas species to C. elegans. At (A) 24 h, (B) 48 h, and (C) 72 h post Aeromonas infection, C. elegans infected with A. dhakensis has the lowest survival rate. The result suggests that A. dhakensis is the most toxic Aeromonas species to C. elegans (***: P &#60; 0.001; **: P &#60; 0.01, Error bars: standard deviation, SD). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58768/58768fig2large.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/58768/58768fig3.jpg" /> 
Figure 3: The corresponding scores for muscle necrosis in C. elegans induced by Aeromonas infection. The criteria for muscle necrosis and corresponding scores are as follows: 3 points for missing muscle fibers; 2 points for ruptured or broken muscle fibers; 1 point for bent muscle fibers; 0 points for healthy muscle fibers. Bent muscle fibers (arrows), ruptured muscle fibers (arrowheads), and missing muscle fibers (star) are indicated. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58768/58768fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/58768/58768fig4.jpg" /> 
Figure 4: The C. elegans muscle necrosis scores. The muscle necrosis scores show the various toxicities of different Aeromonas species to C. elegans. At (A) 24 h, (B) 48 h, and (C) 72 h post Aeromonas infection, the C. elegans muscle damage is the severest from infection with A. dhakensis. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58768/58768fig4large.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>Solution</td>
			<td>Preparation</td>
		</tr>
		<tr>
			<td>Phosphate Buffer</td>
			<td>Dissolve 119.35 g of KH2PO4 and 21.43 g of K2HPO4, in 1 L deionized water. 
			Autoclave at 121 &#176;C for 20 min 
			Adjust the pH value to pH = 6.0 with KOH. 
			Wait until cooled to room temperature</td>
		</tr>
		<tr>
			<td>S Basal</td>
			<td>Dissolve 5.85 g NaCl, 6 g KH2PO4, and 1 g K2HPO4 in 1 L of deionized water. 
			Autoclave at 121 &#176;C for 20 min. 
			Wait until cooled to room temperature.</td>
		</tr>
		<tr>
			<td>1 M Potassium citrate</td>
			<td>Dissolve 4 g citric acid&#8226;H2O and 58.7 g tri-potassium citrate&#8226;H2O in 0.2 mL of deionized water. 
			Adjust the pH value to pH=6.0. 
			Autoclave at 121 &#176;C for 20 min. 
			Wait until cooled to room temperature.</td>
		</tr>
		<tr>
			<td>Trace metals solution</td>
			<td>Dissolve 1.86 g disodium EDTA, 0.69 g FeSO4&#8226;7 H2O, 0.2 g MnCl2&#8226;4 H2O, 0.29 g ZnSO4&#8226;7 H2O, 
			0.025 g CuSO4&#8226;5 H2O in 1 L deionized water. 
			Autoclave at 121 &#176;C for 20 min. 
			Wait until cooled to room temperature 
			Keep the solution in the dark.</td>
		</tr>
		<tr>
			<td>LB broth</td>
			<td>Dissolve 10 g NaCl, 10 g tryptone, and 5 g yeast extract in 1 L of deionized water. Autoclave at 121 &#176;C for 20 min. 
			Wait until cooled to room temperature.</td>
		</tr>
	</tbody>
</table>
Table 1: Solution Preparation.

Watch this Scientific Journal Video about Evaluating Virulence and Pathogenesis of Aeromonas Infection in a Caenorhabditis elegans Model at JoVE.com

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.

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 ...

evaluating-virulence-pathogenesis-aeromonas-infection-caenorhabditis

Nanyang Technological University

Research

JoVE Journal

Immunology and Infection

7.2K Views.  National Cheng Kung University. Deze methode kan helpen bij het beantwoorden van belangrijke vragen in het interactieveld gastheer-ziekteverwekker, zoals de pathogeniteit en virulentie van bacteriële infectie. Het belangrijkste voordeel van deze techniek is dat we de toxiciteit onder en binnen Aeromonas-soorten kunnen evalueren en kunnen bijdragen aan ons begrip van de pathogenese van Aeromonas-infectie. Om te beginnen wassen ongeveer 2.000 aangetrokken volwassen wormen met steriel gedeïmiseerd water.Herhaal de was...

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

The Nematode Caenorhabditis Elegans - A Versatile In Vivo Model to Study Host-microbe Interactions

We demonstrate a method using Caenorhabditis elegans as a model host to study microbial interaction. Microbes are introduced via the diet making the intestine the primary location for disease. The nematode intestine structurally and functionally mimics mammalian intestines and is transparent making it amenable to microscopic study of colonization. Here we show that pathogens can cause disease and death. We are able to identify microbial mutants that show altered virulence. Its conserved innate response to biotic stresses makes C. elegans an excellent system to probe facets of host innate immune interactions. We show that hosts with mutations in the dual oxidase gene cannot produce reactive oxygen species and are unable to resist microbial insult. We further demonstrate the versatility of the presented survival assay by showing that it can be used to study the effects of inhibitors of microbial growth. This assay may also be used to discover fungal virulence factors as targets for the development of novel antifungal agents, as well as provide an opportunity to further uncover host-microbe interactions. The design of this assay lends itself well to high throughput whole-genome screens, while the ability to cryo-preserve worms for future use makes it a cost-effective and attractive whole animal model to study.

The Nematode Caenorhabditis Elegans - A Versatile In Vivo Model to Study Host-microbe Interactions

Here, we present the nematode Caenorhabditis elegans as a versatile host model to study microbial interaction.

We demonstrate a method using Caenorhabditis elegans as a model host to study microbial interaction. Microbes are introduced via the diet making the ...

Genetics

A High-throughput Assay for the Prediction of Chemical Toxicity by Automated Phenotypic Profiling of Caenorhabditis elegans

Applying toxicity testing of chemicals in higher order organisms, such as mice or rats, is time-consuming and expensive, due to their long lifespan and maintenance issues. On the contrary, the nematode Caenorhabditis elegans (C. elegans) has advantages to make it an ideal choice for toxicity testing: a short lifespan, easy cultivation, and efficient reproduction. Here, we describe a protocol for the automatic phenotypic profiling of C. elegans in a 384-well plate. The nematode worms are cultured in a 384-well plate with liquid medium and chemical treatment, and videos are taken of each well to quantify the chemical influence on 33 worm features. Experimental results demonstrate that the quantified phenotype features can classify and predict the acute toxicity for different chemical compounds and establish a priority list for further traditional chemical toxicity assessment tests in a rodent model.

A High-throughput Assay for the Prediction of Chemical Toxicity by Automated Phenotypic Profiling of Caenorhabditis elegans

A quantitative method has been developed to identify and predict the acute toxicity of chemicals by automatically analyzing the phenotypic profiling of Caenorhabditis elegans. This protocol describes how to treat worms with chemicals in a 384-well plate, capture videos, and quantify toxicological related phenotypes.

Applying toxicity testing of chemicals in higher order organisms, such as mice or rats, is time-consuming and expensive, due to their long lifespan and ...

Environment

Two Infection Assays to Study Non-Lethal Virulence Phenotypes in C. Albicans using C. Elegans

While pathogens can be deadly to humans, many of them cause a range of infection types with non-lethal phenotypes. Candida albicans, an opportunistic fungal pathogen of humans, is the fourth most common cause of nosocomial infections which results in ~40% mortality. However, other C. albicans infections are less severe and rarely lethal and include vulvovaginal candidiasis, impacting ~75% of women, as well as oropharyngeal candidiasis, predominantly impacting infants, AIDS patients and cancer patients. While murine models are most frequently used to study C. albicans pathogenesis, these models predominantly assess host survival and are costly, time consuming, and limited in replication. Therefore, several mini-model systems, including Drosophila melanogaster, Danio rerio, Galleria mellonella, and Caenorhabditis elegans, have been developed to study C. albicans. These mini-models are well-suited for screening mutant libraries or diverse genetic backgrounds of C. albicans. Here we describe two approaches to study C. albicans infection using C. elegans. The first is a fecundity assay which measures host reproduction and monitors survival of individual hosts. The second is a lineage expansion assay which measures how C. albicans infection affects host population growth over multiple generations. Together, these assays provide a simple, cost-effective way to quickly assess C. albicans virulence.

Two Infection Assays to Study Non-Lethal Virulence Phenotypes in C. Albicans using C. Elegans

Fungal opportunist pathogens can cause life-threatening as well as minor infections, but non-lethal phenotypes are frequently ignored when studying virulence. Therefore, we developed a nematode model that monitors both the survival and reproduction aspects of host to investigate fungal virulence.

While pathogens can be deadly to humans, many of them cause a range of infection types with non-lethal phenotypes. Candida albicans, an opportunistic ...

Automated Separation of C. elegans Variably Colonized by a Bacterial Pathogen

The wormsorter is an instrument analogous to a FACS machine that is used in studies of Caenorhabditis elegans, typically to sort worms based on expression of a fluorescent reporter. Here, we highlight an alternative usage of this instrument, for sorting worms according to their degree of colonization by a GFP-expressing pathogen. This new usage allowed us to address the relationship between colonization of the worm intestine and induction of immune responses. While C. elegans immune responses to different pathogens have been documented, it is still unknown what initiates them. The two main possibilities (which are not mutually exclusive) are recognition of pathogen-associated molecular patterns, and detection of damage caused by infection. To differentiate between the two possibilities, exposure to the pathogen must be dissociated from the damage it causes. The wormsorter enabled separation of worms that were extensively-colonized by the Gram-negative pathogen Pseudomonas aeruginosa, with the damage likely caused by pathogen load, from worms that were similarly exposed, but not, or marginally, colonized. These distinct populations were used to assess the relationship between pathogen load and the induction of transcriptional immune responses. The results suggest that the two are dissociated, supporting the possibility of pathogen recognition.

Automated Separation of C. elegans Variably Colonized by a Bacterial Pathogen

The wormsorter facilitates genetic screens in Caenorhabditis elegans by sorting worms according to expression of fluorescent reporters. Here, we describe a new usage:&#160;sorting according to colonization by a GFP-expressing pathogen, and we employ it to examine the poorly understood role of pathogen recognition in initiating immune responses.

The wormsorter is an instrument analogous to a FACS machine that is used in studies of Caenorhabditis elegans, typically to sort worms based on expression ...

A High-throughput, High-content, Liquid-based C. elegans Pathosystem

The number of new drugs identified by traditional, in vitro screens has waned, reducing the success of this approach in the search for new weapons to combat multiple drug resistance. This has led to the conclusion that researchers do not only need to find new drugs, but also need to develop new ways of finding them. Amongst the most promising candidate methods are whole-organism, in vivo assays that use high-throughput, phenotypic readouts and hosts that range from Caenorhabditis elegans to Danio rerio. These hosts have several powerful advantages, including dramatic reductions in false positive hits, as compounds that are toxic to the host and/or biounavailable are typically dropped in the initial screen, prior to costly follow up.
Here we show how our assay has been used to interrogate host variation in the well-documented C. elegans&#8212;Pseudomonas aeruginosa liquid killing pathosystem. We also demonstrate several extensions of this well-worked out technique. For example, we are able to carry out high-throughput genetic screens using RNAi in 24- or 96-well plate formats to query host factors in this host-pathogen interaction. Using this assay, whole genome screens can be completed in only a few months, which can dramatically simplify the task of identifying drug targets, potentially without the need for laborious biochemical purification approaches.
We also report here a variation of our method that substitutes the gram-positive bacterium Enterococcus faecalis for the gram-negative pathogen P. aeruginosa. Much as is the case for P. aeruginosa, killing by E. faecalis is time-dependent. Unlike previous C. elegans&#8212;E. faecalis assays, our assay for E. faecalis does not require preinfection, improving its safety profile and reducing the chances of contaminating liquid-handling equipment. The assay is highly robust, showing ~95% death rates 96 h post infection.

A High-throughput, High-content, Liquid-based C. elegans Pathosystem

Here we describe a protocol that is an adaptable, whole host, high-content screening tool that can be utilized to study host-pathogen interactions and be used for drug discovery.

The number of new drugs identified by traditional, in vitro screens has waned, reducing the success of this approach in the search for new weapons to ...

A Caenorhabditis elegans Nutritional-status Based Copper Aversion Assay

To ensure survival, organisms must be capable of avoiding unfavorable habitats while ensuring a consistent food source. Caenorhabditis elegans alter their locomotory patterns upon detection of diverse environmental stimuli and can modulate their suite of behavioral responses in response to starvation conditions. Nematodes typically exhibit a decreased aversive response when removed from a food source for over 30 min. Observation of behavioral changes in response to a changing nutritional status can provide insight into the mechanisms that regulate the transition from a well-fed to starved state.
We have developed an assay that measures a nematode&#39;s ability to cross an aversive barrier (i.e. copper) then reach a food source over a prolonged period of time. This protocol builds upon previous work by integrating multiple variables in a manner that allows for continued data collection as the organisms shift towards an increasingly starved condition. Moreover, this assay permits an increased sample size so that larger populations of nematodes can be simultaneously evaluated.
Organisms defective for the ability to detect or respond to copper immediately cross the chemical barrier, while wild type nematodes are initially repelled. As wild type worms are increasingly starved, they begin to cross the barrier and reach the food source. We designed this assay to evaluate a mutant that is incapable of responding to diverse environmental cues, including food sensation or detection of aversive chemicals. When evaluated via this protocol, the defective organisms immediately crossed the barrier, but were also incapable of detecting a food source. Hence, these mutants repeatedly cross the chemical barrier despite temporarily reaching a food source. This assay can straightforwardly test populations of worms to evaluate potential pathway defects related to aversion and starvation.

A Caenorhabditis elegans Nutritional-status Based Copper Aversion Assay

Here, we present a Caenorhabditis elegans-specific assay designed to evaluate changes in copper aversion behavior and the ability to locate a common food source, as the organism progresses from a well-fed to starved nutritional state.

To ensure survival, organisms must be capable of avoiding unfavorable habitats while ensuring a consistent food source. Caenorhabditis elegans alter their ...

Neuroscience

Selective Cleaning of Wild Caenorhabditis Nematodes to Enrich for Intestinal Microbiome Bacteria

Caenorhabditis elegans (C. elegans)&#160;has proven to be an excellent model for studying host-microbe interactions and the microbiome, especially in the context of the intestines. Recently, ecological sampling of wild Caenorhabditis nematodes has discovered a diverse array of associated microbes, including bacteria, viruses, fungi, and microsporidia. Many of these microbes have interesting colonization or infection phenotypes that warrant further study, but they are often unculturable. This protocol presents a method to enrich the desired intestinal microbes in C. elegans and related nematodes and reduce the presence of the many contaminating microbes adhering to the cuticle. This protocol involves forcing animals into the dauer stage of development and using a series of antibiotic and detergent washes to remove external contamination. As dauer animals have physiological changes that protect nematodes from harsh environmental conditions, any intestinal microbes will be protected from these conditions. But, for enrichment to work, the microbe of interest must be maintained when animals develop into dauers. When the animals leave the dauer stage, they are singly propagated into individual lines. F1 populations are then selected for desired microbes or infection phenotypes and against visible contamination. These methods will allow researchers to enrich unculturable microbes in the intestinal lumen, which make up part of the natural microbiome of C. elegans and intracellular intestinal pathogens. These microbes can then be studied for colonization or infection phenotypes and their effects on the host fitness.

Selective Cleaning of Wild Caenorhabditis Nematodes to Enrich for Intestinal Microbiome Bacteria

Wild Caenorhabditis nematodes are associated with many microbes, often in the gut lumen or infecting the intestine. This protocol details a method to enrich unculturable microbes colonizing the intestine, taking advantage of the resistance of the dauer cuticle.

Caenorhabditis elegans (C. elegans)&#160;has proven to be an excellent model for studying host-microbe interactions and the microbiome, especially in the ...

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.

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.

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

RNA Fluorescence in situ Hybridization (FISH) to Visualize Microbial Colonization and Infection in Caenorhabditis elegans Intestines

The intestines of wild Caenorhabditis nematodes are inhabited by a variety of microorganisms, including gut microbiome bacteria and pathogens, such as microsporidia and viruses. Because of the similarities between Caenorhabditis elegans and mammalian intestinal cells, as well as the power of the C. elegans system, this host has emerged as a model system to study host intestine-microbe interactions in vivo. While it is possible to observe some aspects of these interactions with bright-field microscopy, it is difficult to accurately classify microbes and characterize the extent of colonization or infection without more precise tools. RNA fluorescence in situ hybridization (FISH) can be used as a tool to identify and visualize microbes in nematodes from the wild or to experimentally characterize and quantify infection in nematodes infected with microbes in the lab. FISH probes, labeling the highly abundant small subunit ribosomal RNA, produce a bright signal for bacteria and microsporidian cells. Probes designed to target conserved regions of ribosomal RNA common to many species can detect a broad range of microbes, whereas targeting divergent regions of the ribosomal RNA is useful for narrower detection. Similarly, probes can be designed to label viral RNA. A protocol for RNA FISH staining with either paraformaldehyde (PFA) or acetone fixation is presented. PFA fixation is ideal for nematodes associated with bacteria, microsporidia, and viruses, whereas acetone fixation is necessary for the visualization of microsporida spores. Animals were first washed and fixed in paraformaldehyde or acetone. After fixation, FISH probes were incubated with samples to allow for the hybridization of probes to the desired target. The animals were again washed and then examined on microscope slides or using automated approaches. Overall, this FISH protocol enables detection, identification, and quantification of the microbes that inhabit the C. elegans intestine, including microbes for which there are no genetic tools available.

RNA Fluorescence in situ Hybridization FISH to Visualize Microbial Colonization and Infection in Caenorhabditis elegans Intestines

Intestinal microbes, including extracellular bacteria and intracellular pathogens like the Orsay virus and microsporidia (fungi), are often associated with wild Caenorhabditis nematodes. This article presents a protocol for detecting and quantifying microbes that colonize and/or infect C. elegans nematodes, and for measuring pathogen load after controlled infections in the lab.

The intestines of wild Caenorhabditis nematodes are inhabited by a variety of microorganisms, including gut microbiome bacteria and pathogens, such as ...

Measuring the Effects of Bacteria on C. Elegans Behavior Using an Egg Retention Assay

C. elegans egg-laying behavior is affected by environmental cues such as osmolarity1 and vibration2. In the total absence of food C. elegans also cease egg-laying and retain fertilized eggs in their uterus3. However, the effect of different sources of food, especially pathogenic bacteria and particularly Enterococcus faecalis, on egg-laying
	behavior is not well characterized. The egg-in-worm (EIW) assay is a useful tool to quantify the effects of different types of bacteria, in this case E. faecalis, on egg- laying behavior.
	
	EIW assays involve counting the number of eggs retained in the uterus of C. elegans4. The EIW assay involves bleaching staged, gravid adult C. elegans to remove the cuticle and separate the retained eggs from the animal. Prior to bleaching, worms are exposed to bacteria (or any type of environmental cue) for a fixed period of time. After bleaching, one is very easily able to count the number of eggs retained inside the uterus of the worms. In this assay, a quantifiable increase in egg retention after E. faecalis exposure can be easily measured. The EIW assay is a behavioral assay that may be used to screen for potentially pathogenic bacteria or the presence of environmental toxins. In addition, the EIW assay may be a tool to screen for drugs that affect neurotransmitter signaling since egg-laying behavior is modulated by neurotransmitters such as serotonin and acetylcholine5-9.

Measuring the Effects of Bacteria on C. Elegans Behavior Using an Egg Retention Assay

An egg-in-worm (EIW) assay is a useful method to quantify egg-laying behavior. Alterations in egg laying can be a behavioral response of the model organism Caenorhabditis elegans to potentially harmful environmental substances such as those produced by pathogenic bacteria.

C. elegans egg-laying behavior is  affected by environmental cues such as osmolarity1 and  vibration2. In the total absence of food C. elegans also cease ...

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

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