The work described in this manuscript was supported by NIH grants R01OD024780 and R01AG061302. K.T. was further supported by a Summer Undergraduate Research Fellowship from the University of California, Berkeley, funded by the Rose Hills Foundation. Cartoon designs in Figure 1 were obtained from BioRender.com.

1. Preparation of compost
<ol>
	<li>Obtain compost or garden soil from any convenient source and store inside the laboratory in a standard kitchen plastic container with holes cut in the lid to let air in. Plug the holes with cotton wool to keep fruit flies and other invertebrates out (Figure 1A). 
	NOTE: Five hundred grams of soil (fitting in a 1.5 gallon container, dimensions: 30 cm x 20 cm x 10 cm) will provide enough material for 12 microcosms.</li>
	<li>Enrich the compost or soil with chopped produce or a mixture of different produce in a 1:2 mass ratio of produce to soil.</li>
	<li>Incubate for 7-14 days at 20-25 &#176;C, mixing once a day and adding M9 medium as necessary to maintain moisture without making it muddy. 
	NOTE: Soils not enriched with produce usually do not support C. elegans growth, but which specific produce to use is up to the researcher, with many types and mixes capable of supporting worm growth. Enrichment with different produce will promote bacterial community diversification in different ways, enabling the study of gut microbiome assembly from different starting points (see the discussion section).</li>
</ol>
2. Preparation of compost microcosms
<ol>
	<li>For each microcosm, add 10 g of enriched compost to a 30 mL glass beaker covered with tin foil and autoclave (Figure 1B).</li>
	<li>To prepare microbial extract to replenish the autoclaved compost, start by adding 30 g of the same compost used in step 2.1 to each of three 50 mL tubes and fill with M9. Vortex for 1 min (Figure 1C). 
	NOTE: This should provide enough bacteria for nine microcosms, each supporting the development of hundreds of nematodes.</li>
	<li>Centrifuge the tubes at 560 &#215; g for 5 min at room temperature (RT).</li>
	<li>Paying attention not to disturb the pellet, remove the supernatants with a serological pipette and combine in a new 50 mL tube.</li>
	<li>Concentrate the bacterial extract by centrifuging at maximum speed (2,000 &#215; g) for 15 min at RT. Resuspend the pellets in enough M9 to have 200 &#181;L for each microcosm and 200 &#181;L more to add to a plate that will serve as a visible proxy of worm development inside microcosms. 
	NOTE: For example, for nine microcosms, resuspend the microbial pellet in 2 mL of M9.</li>
	<li>Add 200 &#181;L of the concentrated microbial extract to each beaker of autoclaved compost, as well as to an NGM plate that will serve as the visible proxy plate.</li>
	<li>Incubate microcosms and proxy plate for 24 h at 20-25 &#176;C before addition of worms.</li>
</ol>
3. Raising worms in compost microcosms
<ol>
	<li>Add 500-1000 eggs or L1 larvae3 to each microcosm and to the proxy plate (step 2.7) to begin the experiment (Figure 1D). 
	&#8203;NOTE: The experiments described here utilize N2 wild-type worms. However, other C. elegans strains (and potentially other nematodes) can be used.</li>
	<li>Raise the worms to adulthood at 20 &#176;C (typically 3 days).</li>
</ol>
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/64393/64393fig01.jpg" /> 
Figure 1: Preparing compost microcosms, raising worms, and harvesting. (A) Enrich local soil or compost with produce and incubate for 2 weeks. Combine (B) autoclaved enriched soil with (C) microbial extract and (D) incubate for at least 24 h before adding synchronized L1s worms to microcosms to begin the experiment. (E, F) When ready to harvest, add compost from the microcosm into a Baermann funnel support cylinder and cover with M9. (G) After 15 min, release the filtrate into a 50 mL tube. Abbreviation: sup. = supernatant. <a href="https://www.jove.com/files/ftp_upload/64393/64393fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
4. Preparing a Baermann funnel for harvesting worms
<ol>
	<li>Assemble a Baermann funnel by attaching 5-8 cm of rigid rubber tubing to the end of a plastic funnel.</li>
	<li>Slide a clamp onto the tubing and clamp it shut.</li>
	<li>Place in the funnel a 7 cm long, 5 cm diameter, cylindrical&#160;PVC pipe with a 1 mm nylon mesh glued at its bottom (Figure 1E). Line the cylinder with two sheets of tissue paper.</li>
	<li>Place the Baermann funnel into a flask (Figure 1F).</li>
</ol>
5. Harvesting worms from microcosms and collecting the respective soil samples
<ol>
	<li>Add 20 mL of M9 to the microcosm in which the worms were raised, agitate the mixture, and then pour the mixture from the beaker into the tissue paper-lined cylinder in the Baermann funnel setup. Add more M9 to completely submerge the compost in the funnel. 
	NOTE: C. elegans (N2) populations reach adulthood in compost microcosms at a similar rate as on standard agar plates seeded with Escherichia coli. For further accuracy in harvesting worms at a specific developmental stage, refer to the proxy plate from step 3.3.</li>
	<li>After 30 min, unfasten the clamp to release the filtrate containing the harvested worms into a 50 mL tube (Figure 1G).</li>
	<li>Add more M9 to the cylinder and repeat for a second round to harvest more worms, and once more if additional worms are required. 
	NOTE: When repeating harvesting rounds, be careful not to compromise the tissue paper&#39;s integrity, which could allow soil particles to pass through. A maximum of four harvest rounds should isolate at least 50% of the worms originally added to the microcosm without compromising tissue paper integrity.</li>
	<li>Concentrate the worms by centrifuging at 560 &#215; g for 2 min (RT). Remove 35 mL of the supernatant with a serological pipette.</li>
	<li>Transfer the remaining 15 mL to a 15 mL tube and centrifuge again at 560 &#215; g for 1 min to further concentrate the worms. Remove 14 mL of the supernatant with a serological pipette.</li>
	<li>In parallel, collect 1 g of the remaining microcosm soil into a 1.5 mL tube. Process the soil samples containing the environmental bacterial community immediately or store them at &#8722;20 &#176;C for later extraction of nucleic acids, as described below for worm samples.</li>
</ol>
6. Washing and surface sterilization of harvested worms
<ol>
	<li>Transfer 1 mL of the concentrated, harvested worms from step 5.5 to a 1.5 mL tube using a glass pipette. Incubate for 2 min to allow the worms to settle at the bottom of the tube. Remove the supernatant, leaving the bottom 100 &#181;L undisturbed.</li>
	<li>Wash 6x with 1.5 mL of M9+T (0.025% Triton-X in M9), allowing the worms to settle at the bottom each time.</li>
	<li>Transfer the washed worms in a volume of 100 &#181;L to a new 1.5 mL tube using a glass pipette. 
	NOTE: At this point in the protocol, roughly 30 min should have passed since the first wash (step 6.2). Allowing the worms to remain without food for at least 1 h before the addition of levamisole is recommended to allow the excretion of transient bacteria and the full digestion of food bacteria.</li>
	<li>Add 100 &#181;L of 25 mM levamisole hydrochloride to paralyze the worms. Incubate for 5 min at RT.</li>
	<li>Add 200 &#181;L of 4% bleach solution. Incubate for 2 min.</li>
	<li>Remove the supernatant, leaving the bottom-most 150 &#181;L undisturbed, and wash 3x with M9+T, as above. 
	NOTE: To minimize contamination of the samples, it is recommended to use filtered M9+T (through a 0.2 &#181;m filter) and to wear gloves from this point onward.</li>
	<li>Following the washes, take 50 &#181;L of the remaining 150 &#181;L from the last wash and plate on an LB plate, incubating at 25 &#176;C for 48 h to confirm the effective removal of external bacteria. 
	NOTE: Observing up to 30 colonies in the last wash (representing 60 external bacterial cells total remaining in the sample) is permitted and expected to have only a marginal contribution to the analyzed microbiome composition, given the thousands of bacteria that typically colonize each adult worm15. When more are observed, sample integrity might be compromised.</li>
	<li>Use the surface-sterilized worms immediately (e.g., to extract live bacteria for culturing [CFU counts]) or store at &#8722;20 &#176;C for the subsequent extraction of nucleic acids.</li>
</ol>
7. DNA extraction
NOTE: The following steps describe DNA extraction of the harvested worms using a commercial kit designed for the extraction of microbial DNA from soil (see Table of Materials), with modifications described below to facilitate the extraction of microbial DNA from worms.
<ol>
	<li>Transfer the sample containing surface-sterilized worms (defrost at RT if necessary) to the tubes provided by the kit, replacing the provided glass beads with roughly 30-50 1 mm diameter zirconia beads (see Table of Materials). 
	NOTE: The observed DNA yields are higher with zirconia beads than with the glass beads provided by the kit. Although the reason behind this observation remains undetermined, zirconia beads may break open the nematode cuticle more efficiently, releasing more bacteria.</li>
	<li>For compost samples, add roughly 250 mg of the collected soil to the kit&#39;s tubes without replacing the glass beads.</li>
	<li>After the addition of the buffer solution provided by the kit to all the samples, homogenize with a power homogenizer at RT (see Table of Materials) for two rounds of 2,000 rpm for 30 s each, pausing for 30 s in between.</li>
	<li>Complete the remaining DNA purification steps according to the kit protocol. Elute the worm samples in no more than 50 &#181;L of elution buffer to ensure DNA concentrations that are high enough for sequencing.</li>
</ol>

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M., Ratzke, C., Gore, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interspecies+bacterial+competition+regulates+community+assembly+in+the+C.+elegans+intestine.">Interspecies bacterial competition regulates community assembly in the C. elegans intestine.</a> ISME Journal. 15 (7), 2131-2145 (2021).</li><li>Berg, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=TGF&#946;/BMP+immune+signaling+affects+abundance+and+function+of+C.+elegans+gut+commensals.">TGF&#946;/BMP immune signaling affects abundance and function of C. elegans gut commensals.</a> Nature Communications. 10, 604 (2019).</li><li>Zhang, F., 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=Natural+genetic+variation+drives+microbiome+selection+in+the+Caenorhabditis+elegans+gut.">Natural genetic variation drives microbiome selection in the Caenorhabditis elegans gut.</a> Current Biology. 31 (12), 2603-2618 (2021).</li><li>Montalvo-Katz, S., Huang, H., Appel, M. D., Berg, M., Shapira, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Association+with+soil+bacteria+enhances+p38-dependent+infection+resistance+in+Caenorhabditis+elegans.">Association with soil bacteria enhances p38-dependent infection resistance in Caenorhabditis elegans.</a> Infection and Immunity. 81 (2), 514-520 (2013).</li><li>Fr&#233;zal, L., F&#233;lix, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=C.+elegans+outside+the+Petri+dish.">C. elegans outside the Petri dish.</a> eLife. 4, 05849 (2015).</li><li>Dirksen, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+native+microbiome+of+the+nematode+Caenorhabditis+elegans:+Gateway+to+a+new+host-microbiome+model.">The native microbiome of the nematode Caenorhabditis elegans: Gateway to a new host-microbiome model.</a> BMC Biology. 14 (1), 38 (2016).</li><li>Samuel, B. S., Rowedder, H., Braendle, C., F&#233;lix, M. A., Ruvkun, G. <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+responses+to+bacteria+from+its+natural+habitats.">Caenorhabditis elegans responses to bacteria from its natural habitats.</a> Proceedings of the National Academy of Sciences of the United States of America. 113 (27), 3941-3949 (2016).</li><li>Berg, M., Zhou, X. Y., Shapira, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Host-specific+functional+significance+of+Caenorhabditis+gut+commensals.">Host-specific functional significance of Caenorhabditis gut commensals.</a> Frontiers in Microbiology. 7, 1622 (2016).</li><li>Berg, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assembly+of+the+Caenorhabditis+elegans+gut+microbiota+from+diverse+soil+microbial+environments.">Assembly of the Caenorhabditis elegans gut microbiota from diverse soil microbial environments.</a> ISME Journal. 10 (8), 1998-2009 (2016).</li><li>Slowinski, S., 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=Interactions+with+a+complex+microbiota+mediate+a+trade-off+between+the+host+development+rate+and+heat+stress+resistance.">Interactions with a complex microbiota mediate a trade-off between the host development rate and heat stress resistance.</a> Microorganisms. 8 (11), 1-9 (2020).</li><li>P&#233;rez-Carrascal, O. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Host+preference+of+beneficial+commensals+in+a+microbially-diverse+environment.">Host preference of beneficial commensals in a microbially-diverse environment.</a> Frontiers in Cellular and Infection Microbiology. 12, 795343 (2022).</li><li>Callahan, B. 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=DADA2:+High-resolution+sample+inference+from+Illumina+amplicon+data.">DADA2: High-resolution sample inference from Illumina amplicon data.</a> Nature Methods. 13 (7), 581-583 (2016).</li><li>Quast, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+SILVA+ribosomal+RNA+gene+database+project:+Improved+data+processing+and+web-based+tools.">The SILVA ribosomal RNA gene database project: Improved data processing and web-based tools.</a> Nucleic Acids Research. 41, 590-596 (2013).</li><li>McMurdie, P. J., Holmes, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=phyloseq:+An+R+package+for+reproducible+interactive+analysis+and+graphics+of+microbiome+census+data.">phyloseq: An R package for reproducible interactive analysis and graphics of microbiome census data.</a> PLoS One. 8 (4), 61217 (2013).</li><li>Lozupone, C., Knight, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=UniFrac:+A+new+phylogenetic+method+for+comparing+microbial+communities.">UniFrac: A new phylogenetic method for comparing microbial communities.</a> Applied and Environmental Microbiology. 71 (12), 8228-8235 (2005).</li><li>Lozupone, C., Lladser, M. E., Knights, D., Stombaugh, J., Knight, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=UniFrac:+An+effective+distance+metric+for+microbial+community+comparison.">UniFrac: An effective distance metric for microbial community comparison.</a> ISME Journal. 5 (2), 169-172 (2011).</li></ol>

The authors have no conflicts of interest to declare that are relevant to the content of this article.

The protocol presented here describes a method to study the gut microbiome of nematodes raised in natural-like environments, offering an alternative approach to the isolation of worms from nature or to raising them on synthetic communities.
The thousands of potential bacterial species captured in the representative microcosm experiment reflect the microbial diversity that the worms have evolved with and demonstrate the ability of the microcosm pipeline to combine the advantages of working with a model host organism and those of working with natural, diverse microbial communities.
The representative results demonstrate that enriching a common soil with different produce types modulates environmental microbial diversity, highlighting the range of microbial diversity available for exploration using this pipeline. Produce choice is not particularly important. Previous work has used bananas, apples, oranges, strawberries, green tea leaves, and potatoes to enrich soil, resulting in similar worm raising efficiency. Mixed produce has also been used effectively. The key feature is that different produce will diversify a given soil in distinct ways.
Despite the broad variation in environmental microbial diversity, worm gut microbiomes vary considerably less, recapitulating the importance of the worm gut niche and host filtering for the assembly of a core gut microbiome that is distinct from that of its soil environment13. This pattern is observed best with weighted UniFrac PCoA analysis, demonstrating that the differences between environmental soil and worm gut microbiomes stem mostly from differences in the relative abundance of key taxa.
Although the protocol described here focuses on harvesting worms for 16S sequencing, microcosms can be used to explore additional questions of interest. For example, worms harvested from microcosms can be subsequently examined for the effect of a diverse gut microbiome on host resistance to various adverse conditions, including pathogens or toxins. Alternatively, novel bacterial species and strains may be isolated and cultured from ground-harvested worms, expanding the taxonomic and functional diversity of bacteria available to perform experiments with.
While research methods in laboratory settings seek consistency and reproducibility, work with microcosms takes advantage of the natural variation to explore host-microbe interactions in a natural-like context. Nevertheless, this variation also poses some challenges. Some soils that include high levels of endogenous invertebrates may require additional centrifugation, filtration, and examination steps to effectively eliminate undesired organisms from the microcosm preparation. Additionally, low microbial abundance in soil may induce undesired dauer formation in worm populations, requiring an increase in microbial extract or enriching soil with a greater amount of produce. With each microcosm experiment performed, researchers continue to explore the full taxonomic and functional diversity provided by nature, enabling the discovery of new microbial taxa and functional abilities ranging from infection resistance to protection against environmental xenobiotics.

The nematode Caenorhabditis elegans is emerging as a useful model for studying interactions between hosts and their gut microbiomes1,2. As a model, it offers several advantages. First, germ-free or gnotobiotic animals are easy to obtain and maintain; bleach can be used to kill gravid worms and associated microbes, leaving their bleach-resistant eggs unharmed to grow as age-synchronized populations that can be colonized by bacteria of interest3,4. In addition, when grown in the presence of bacteria, C. elegans, a bacterivore, ingest the encountered bacteria, with susceptible species digested or excreted, while resistant and persistent species stably colonize the worm gut. Furthermore, C. elegans are mostly hermaphroditic, producing populations of genetically identical progeny, which reduces confounding genetic variation. Coupled with the availability of mutant and transgenic worm strains, working with C. elegans offers researchers a gnotobiotic and genetically tractable model to investigate the molecular underpinnings of host-microbe interactions5,6,7,8.
While experiments with well-characterized bacteria can facilitate the analysis of molecular mechanisms, identifying and studying the bacteria that worms interact with in nature are essential for exploring the diversity of such mechanisms, unraveling the natural context for their function, and understanding the selective forces that have shaped their evolution. Outside the laboratory, C. elegans is found globally in humid temperate climates, where populations are thought to undergo a &#34;boom-and-bust&#34; lifecycle, characterized by rapid population growth when resources are abundant, followed by a developmental shift to pioneering, stress-tolerant dauers when resources are depleted9. Though considered a soil nematode, wild-proliferating C. elegans populations are most commonly found feeding on decomposing organic material such as rotting flowers or fruits, where bacterial populations are abundant and diverse.
Studies of the gut microbiome in nematodes isolated from the wild have identified diverse, yet characteristic, bacterial communities10,11, the composition of which was further supported by studies carried out with worms raised in natural-like microcosm environments12,13. Together, such studies enabled the delineation of a core worm gut microbiome2. Whereas the sampling of C. elegans populations in the wild represents the most direct examination of natural worm-microbe interactions, it is not feasible everywhere and anytime, as it is limited to regions and seasons with ample precipitation10,11. Alternatively, instead of isolating worms from their natural habitat, experiments using microcosms bring the natural habitat into the laboratory6,8,12,13,14,15. Microcosm environments are prepared from soil composted with various fruit or vegetables, which enables further diversification of the starting soil community. They offer tractable experimental methods that combine the microbial diversity and the three-dimensional wild soil environment with the experimental advantages of a controlled laboratory facility and genetically defined worm strains. The protocol below details the steps involved in working with compost microcosms, demonstrating their use in understanding the assembly of a characteristic worm gut microbiome from diverse environments.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>AMPure XP Reagent, 60 mL</td>
			<td>Beckman Coulter</td>
			<td>A63881</td>
			<td>Supplementary File Step 1.3</td>
		</tr>
		<tr>
			<td>Bleach (Sodium Hypochlorite)</td>
			<td>Sigma-Aldrich</td>
			<td>7681-52-9</td>
			<td>Step 6.5</td>
		</tr>
		<tr>
			<td>DNeasy PowerSoil Pro Kit</td>
			<td>Qiagen</td>
			<td>47016</td>
			<td>Step 7 DNA extractions</td>
		</tr>
		<tr>
			<td>dNTP set 10 mM</td>
			<td>Invitrogen</td>
			<td>18427013</td>
			<td>Supplementary Step 1.2</td>
		</tr>
		<tr>
			<td>Easypet 3 Serological Pipette Controller</td>
			<td>Eppendorf</td>
			<td>4430000018</td>
			<td>Used to remove supernatant when specified</td>
		</tr>
		<tr>
			<td>Greiner Bio-One 25 mL Sterile Serological Pipets</td>
			<td>Fisher Scientific</td>
			<td>07-000-368</td>
			<td>Used to remove supernatant when specified</td>
		</tr>
		<tr>
			<td>KH2PO4</td>
			<td>Fisher Scientific</td>
			<td>P285-500</td>
			<td>Used to make M9</td>
		</tr>
		<tr>
			<td>Levamisole Hydrochloride</td>
			<td>Fisher Scientific</td>
			<td>AC187870100</td>
			<td>Step 6.4</td>
		</tr>
		<tr>
			<td>M9 Minimal Media Solution</td>
			<td>Prepared in-house</td>
			<td>N/A</td>
			<td>Recipe in wormbook.org</td>
		</tr>
		<tr>
			<td>MgSO4</td>
			<td>Fisher Scientific</td>
			<td>M63-500</td>
			<td>Used to make M9</td>
		</tr>
		<tr>
			<td>MiniSeq High Output Reagent Kit (150 cycles)</td>
			<td>Illumina</td>
			<td>FC-420-1002</td>
			<td>Supplementary Step 1.7</td>
		</tr>
		<tr>
			<td>MiniSeq System</td>
			<td>Illumina</td>
			<td>SY-420-1001</td>
			<td>Commercial sequencer used; Supplementary Step 1.7</td>
		</tr>
		<tr>
			<td>Na2HPO4</td>
			<td>Fisher Scientific</td>
			<td>S374-500</td>
			<td>Used to make M9</td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Fisher Scientific</td>
			<td>S271-3</td>
			<td>Used to make M9</td>
		</tr>
		<tr>
			<td>Nematode Growth Media (NGM)</td>
			<td>Prepared in-house</td>
			<td>N/A</td>
			<td>Recipe in wormbook.org</td>
		</tr>
		<tr>
			<td>Nextera XT DNA Library Preparation Kit (96 samples)</td>
			<td>Illumina</td>
			<td>FC-131-1096</td>
			<td>Library prep kit used; Supplementary Step 1.4</td>
		</tr>
		<tr>
			<td>PhiX Control v3</td>
			<td>Illumina</td>
			<td>FC-110-3001</td>
			<td>Supplementary Step 1.7</td>
		</tr>
		<tr>
			<td>Phusion High-Fidelity DNA Polymerase</td>
			<td>New England Biolabs</td>
			<td>M0530L</td>
			<td>Supplementary Step 1.2</td>
		</tr>
		<tr>
			<td>PowerLyzer 24 Homogenizer (110/220 V)</td>
			<td>Qiagen</td>
			<td>13155</td>
			<td>Step 7.3</td>
		</tr>
		<tr>
			<td>Qubit dsDNA HS Assay Kit</td>
			<td>Invitrogen</td>
			<td>Q32851</td>
			<td>Supplementary Steps 1.1 &#38; 1.6</td>
		</tr>
		<tr>
			<td>Qubit Fluorometer</td>
			<td>Invitrogen</td>
			<td>Q33238</td>
			<td>Supplementary Steps 1.1 &#38; 1.6</td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Fisher Scientific</td>
			<td>BP-151</td>
			<td>Used to prepare M9+T</td>
		</tr>
		<tr>
			<td>Zirconia/Silica Beads 1.0 mm diameter</td>
			<td>Fisher Scientific</td>
			<td>NC9847287</td>
			<td>Step 7.1</td>
		</tr>
	</tbody>
</table>

compost microcosms as microbially diverse, natural-like environments for microbiome research in caenorhabditis elegans

The nematode Caenorhabditis elegans is emerging as a useful model for studying the molecular mechanisms underlying interactions between hosts and their gut microbiomes. While experiments with well-characterized bacteria or defined bacterial communities can facilitate the analysis of molecular mechanisms, studying nematodes in their natural microbial context is essential for exploring the diversity of such mechanisms. At the same time, the isolation of worms from the wild is not always feasible, and, even when possible, sampling from the wild restricts the use of the genetic toolkit otherwise available for C. elegans research. The following protocol describes a method for microbiome studies utilizing compost microcosms for the in-lab&#160;growth in microbially diverse and natural-like environments.
Locally sourced soil can be enriched with produce to diversify the microbial communities in which worms are raised and from which they are harvested, washed, and surface-sterilized for subsequent analyses. Representative experiments demonstrate the ability to modulate the microbial community in a common soil by enriching it with different produce and further demonstrate that worms raised in these distinct environments assemble similar gut microbiomes distinct from their respective environments, supporting the notion of a species-specific core gut microbiome. Overall, compost microcosms provide natural-like in-lab environments for microbiome research as an alternative to synthetic microbial communities or to the isolation of wild nematodes.

To explore the ability to diversify the community of soil microcosms, we compared the microbial communities in compost microcosms prepared by enriching the same initial soil, an industrial-grade compost available from the city of Berkeley, California, with different produce: apples, bell peppers, oranges, or potatoes (each set in triplicate). We further compared the microbial communities of each compost environment with the gut microbiome of wild-type C. elegans raised in the respective microcosm. Analysis was performed with DNA samples extracted from roughly 500 surface-sterilized adults per microcosm and from 250 mg compost samples of the respective microcosms.
Characterization of the environmental soil and worm gut microbiomes relied on next-generation sequencing of the V4 region of the bacterial 16S rRNA gene. Sequencing library preparation was achieved using the standard kits and performed according to the manufacturers&#39; instructions, with sequencing performed on a commercial sequencer (see Table of Materials). Demultiplexed sequences were processed using DADA2, assigned taxonomy based on the SILVA v132 reference database, and analyzed with phyloseq16,17,18&#160;(see Supplementary File 1, Supplementary Figure S1, Supplementary Figure S2, Supplementary Figure S3, Supplementary Table S1, and Supplementary Table S2 for a detailed description of the sequencing and analysis; the full computational pipeline is available in GitHub [https://github.com/kennytrang/CompostMicrocosms]). Raw data are available at the NCBI Sequence Read Archive (Bioproject ID PRJNA856419).
On average, 73,220 sequences were obtained per sample. These sequences represent 15,027 amplicon sequence variants (ASVs), spanning 27 phyla and 216 families, including families considered part of the core C. elegans gut microbiome13, such as Rhizobiaceae, Burkholderiaceae, and Bacillaceae. Enterobacteriaceae, and Pseudomonadaceae, which were previously found to be dominant members, were a minority this time, but were still enriched (2-10-fold) compared to their respective soil environments. Comparisons based on both unweighted and weighted UniFrac19,20 distances demonstrated good reproducibility among microcosm triplicates enriched with the same produce, as indicated by close clustering. In contrast, environmental soil microbiomes enriched with different produce clustered away from each other, demonstrating the ability to diversify an initial microbial community through the addition of different produce (Figure 2).
In comparisons of worm gut microbiomes and environmental communities, principal coordinate analysis (PCoA) with either unweighted or weighted UniFrac distances showed distinct clustering of worm gut microbiomes away from that of their respective environments for each microcosm type (Figure 2). While PCoA based on unweighted UniFrac distances did not distinguish between soil and worm microbiomes (Figure 2A), clustering based on weighted distances revealed a clear separation of worm gut and compost microbiomes (Figure 2B). These results support a process in which host filtering operates on environmental availability to shape a gut microbiome that is not completely distinct from its environmental source with regard to the presence of taxa but modulates their abundance by enriching for a subset of the available taxa, ultimately resulting in a core worm gut microbiome shared between worms raised in different environments.
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/64393/64393fig02.jpg" /> 
Figure 2: Worm gut microbiomes clustering away from their respective produce-diversified microbial environments. Microbiome composition was determined with 16S sequencing, and communities from microcosms enriched with the designated produce or from worms raised in them were clustered using PCoA based on (A) unweighted or (B) weighted UniFrac distances. Axes shown are those that explain the greatest variation in community composition between samples (N = 3 for each microcosm type). <a href="https://www.jove.com/files/ftp_upload/64393/64393fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Supplementary File 1: Next-generation sequencing and data analysis. Presented here are the steps for library preparation, in-lab sequencing, and data analysis. <a href="https://www.jove.com/files/ftp_upload/64393/Supplementary File 1.pdf" target="_blank">Please click here to download this File.</a>
Supplementary Figure S1: An example of a quality control graph for the reverse reads from one sample. The X-axis (cycle) shows the nucleotide position along the sequence read. The left Y-axis shows the quality score. The greyscale heatmap represents the frequency of the quality score at each nucleotide position; the green line depicts the median quality score at each nucleotide position; the top orange line depicts the quartiles of the quality score distribution; the bottom red line depicts the percent of sequence reads that extended that nucleotide position (right Y-axis, here 100%). <a href="https://www.jove.com/files/ftp_upload/64393/Supplementary Figure S1.pdf" target="_blank">Please click here to download this File.</a>
Supplementary Figure S2: Error rates for different samples. The error frequency in the different samples (black dots) should decrease with increasing quality score for each possible base pair substitution depicted, reflecting the expected trend. <a href="https://www.jove.com/files/ftp_upload/64393/Supplementary Figure S2.pdf" target="_blank">Please click here to download this File.</a>
Supplementary Figure S3: An example of PCoA based on weighted UniFrac distances. The group names shown in&#160;the&#160;legend represent the produce used to enrich the compost used in the different microcosms. <a href="https://www.jove.com/files/ftp_upload/64393/Supplementary Figure S3.pdf" target="_blank">Please click here to download this File.</a>
Supplementary Table S1: Sequential sequence filtering. aNumber of sequence reads before filtering. b-dEach column represents the number of sequence reads remaining after a filtration step: filtering out low quality reads (step 2.5), denoising algorithm performed by dada() (step 2.8), merging forward and reverse reads (step 2.9), and removing chimeras (step 2.11). <a href="https://www.jove.com/files/ftp_upload/64393/64393_S1.xlsx" target="_blank">Please click here to download this File.</a>
Supplementary Table S2: Metadata table. <a href="https://www.jove.com/files/ftp_upload/64393/64393_S2.xlsx" target="_blank">Please click here to download this File.</a>

Watch this Scientific Journal Video about Compost Microcosms as Microbially Diverse, Natural-like Environments for Microbiome Research in Caenorhabditis elegans at JoVE.com

Compost Microcosms as Microbially Diverse, Natural-like Environments for Microbiome Research in Caenorhabditis elegans

Compost microcosms bring the microbial diversity found in nature into the laboratory to facilitate microbiome research in Caenorhabditis elegans. Provided here are protocols for setting up microcosm experiments, with the experiments demonstrating the ability to modulate environmental microbial diversity to explore the relationships between environmental microbial diversity and worm gut microbiome composition.

Compost Microcosms as Microbially Diverse, Natural-like Environments for Microbiome Research in Caenorhabditis elegans

The nematode Caenorhabditis elegans is emerging as a useful model for studying the molecular mechanisms underlying interactions between hosts and their ...

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2.1K Views.  University of California, Berkeley. Compost microcosms bring the microbial diversity found in nature into the laboratory to facilitate microbiome research in Caenorhabditis elegans. Provided here are protocols for setting up microcosm experiments, with the experiments demonstrating the ability to modulate environmental microbial diversity to explore the relationships between environmental microbial diversity and worm gut microbiome composition.

Video: Compost Microcosms as Microbially Diverse, Natural-like Environments for Microbiome Research in Caenorhabditis elegans

Measuring Caenorhabditis elegans Life Span in 96 Well Microtiter Plates

Lifespan is a biological process regulated by several genetic pathways. One strategy to investigate the biology of aging is to study animals that harbor mutations in components of age-regulatory pathways. If these mutations perturb the function of the age-regulatory pathway and therefore alter the lifespan of the entire organism, they provide important mechanistic insights1-3.
Another strategy to investigate the regulation of lifespan is to use small molecules to perturb age-regulatory pathways. To date, a number of molecules are known to extend lifespan in various model organisms and are used as tools to study the biology of aging4-16. The number of molecules identified thus far is small compared to the genetic "toolset" that is available to study the biology of aging.
Caenorhabditis elegans is one of the principle models used to study aging because of its excellent genetics and short lifespan of three weeks. More recently, C.elegans has emerged as a model organism for phenotype based drug screens5,7,16-20 because of its small size and its ability to grow in microtiter plates.
Here we present an assay to measure C.elegans lifespan in 96 well microtiter plates. The assay was developed and successfully used to screen large libraries for molecules that extend C.elegans lifespan7. The reliability of the assay was evaluated in multiple tests: first, by measuring the lifespan of wild type animals grown at different temperatures; second, by measuring the lifespan of mutants with altered lifespans; third, by measuring changes in lifespan in response to different concentrations of the antidepressant Mirtazepine. Mirtazepine has previously been shown to extend lifespan in C.elegans7. The results of these tests show that the assay is able to replicate previous findings from other assays and is quantitative. The microtiter format also makes this lifespan assay compatible with automated liquid handling systems and allows integration into automated platforms.

Measuring Caenorhabditis elegans Life Span in 96 Well Microtiter Plates

In this protocol we present a method to measure Caenorhabditis elegans lifespan in 96 well microtiter plates.

Lifespan is a biological process regulated by several genetic pathways. One strategy to investigate the biology of aging is to study animals that harbor ...

Time-lapse Microscopy of Early Embryogenesis in Caenorhabditis elegans

Caenorhabditis elegans has often been used as a model system in studies of early developmental processes. The transparency of the 
embryos, the genetic resources, and the relative ease of transformation are qualities that make C. elegans an excellent model for early embryogenesis. Laser-based 
confocal microscopy and fluorescently labeled tags allow researchers to follow specific cellular structures and proteins in the developing embryo. For example, 
one can follow specific organelles, such as lysosomes or mitochondria, using fluorescently labeled dyes. These dyes can be delivered to the early embryo by means 
of microinjection into the adult gonad. Also, the localization of specific proteins can be followed using fluorescent protein tags. Examples are presented here 
demonstrating the use of a fluorescent lysosomal dye as well as fluorescently tagged histone and ubiquitin proteins. The labeled histone is used to visualize 
the DNA and thus identify the stage of the cell cycle. GFP-tagged ubiquitin reveals the dynamics of ubiquitinated vesicles in the early embryo. Observations 
of labeled lysosomes and GFP:: ubiquitin can be used to determine if there is colocalization between ubiquitinated vesicles and lysosomes. A technique for 
the microinjection of the lysosomal dye is presented. Techniques for generating transgenenic strains are presented elsewhere (1, 2). For imaging, embryos 
are cut out of adult hermaphrodite nematodes and mounted onto 2% agarose pads followed by time-lapse microscopy on a standard laser scanning confocal 
microscope or a spinning disk confocal microscope. This methodology provides for the high resolution visualization of early embryogenesis.

Time-lapse Microscopy of Early Embryogenesis in Caenorhabditis elegans

This article describes a technique for the visualization of the early events of embryogenesis in the nematode Caenorhabditis elegans.

Caenorhabditis elegans has often been used as a model system in studies of early developmental processes.  The transparency of the 
embryos, the genetic ...

Basic Caenorhabditis elegans Methods: Synchronization and Observation

Research into the molecular and developmental biology of the nematode Caenorhabditis elegans was begun in the early seventies by Sydney Brenner and it has since been used extensively as a model organism 1. C. elegans possesses key attributes such as simplicity, transparency and short life cycle that have made it a suitable experimental system for fundamental biological studies for many years 2. Discoveries in this nematode have broad implications because many cellular and molecular processes that control animal development are evolutionary conserved 3.
C. elegans life cycle goes through an embryonic stage and four larval stages before animals reach adulthood. Development can take 2 to 4 days depending on the temperature. In each of the stages several characteristic traits can be observed. The knowledge of its complete cell lineage 4,5 together with the deep annotation of its genome turn this nematode into a great model in fields as diverse as the neurobiology 6, aging 7,8, stem cell biology 9 and germ line biology 10. 
An additional feature that makes C. elegans an attractive model to work with is the possibility of obtaining populations of worms synchronized at a specific stage through a relatively easy protocol. The ease of maintaining and propagating this nematode added to the possibility of synchronization provide a powerful tool to obtain large amounts of worms, which can be used for a wide variety of small or high-throughput experiments such as RNAi screens, microarrays, massive sequencing, immunoblot or in situ hybridization, among others. 
Because of its transparency, C. elegans structures can be distinguished under the microscope using Differential Interference Contrast microscopy, also known as Nomarski microscopy. The use of a fluorescent DNA binder, DAPI (4',6-diamidino-2-phenylindole), for instance, can lead to the specific identification and localization of individual cells, as well as subcellular structures/defects associated to them.

Basic Caenorhabditis elegans Methods: Synchronization and Observation

The easiness of maintaining and propagating the nematode C. elegans make it a nice model organism to work with. The possibility of synchronizing worms allows the work with a significant amount of subjects at the same developmental stage, what facilitates the study of one particular process in many animals.

Research into the molecular and developmental biology of the nematode Caenorhabditis elegans was begun in the early seventies by Sydney Brenner and it has ...

Using Caenorhabditis elegans as a Model System to Study Protein Homeostasis in a Multicellular Organism

The folding and assembly of proteins is essential for protein function, the long-term health of the cell, and longevity of the organism. Historically, the function and regulation of protein folding was studied in vitro, in isolated tissue culture cells and in unicellular organisms. Recent studies have uncovered links between protein homeostasis (proteostasis), metabolism, development, aging, and temperature-sensing. These findings have led to the development of new tools for monitoring protein folding in the model metazoan organism Caenorhabditis elegans. In our laboratory, we combine behavioral assays, imaging and biochemical approaches using temperature-sensitive or naturally occurring metastable proteins as sensors of the folding environment to monitor protein misfolding. Behavioral assays that are associated with the misfolding of a specific protein provide a simple and powerful readout for protein folding, allowing for the fast screening of genes and conditions that modulate folding. Likewise, such misfolding can be associated with protein mislocalization in the cell. Monitoring protein localization can, therefore, highlight changes in cellular folding capacity occurring in different tissues, at various stages of development and in the face of changing conditions. Finally, using biochemical tools ex vivo, we can directly monitor protein stability and conformation. Thus, by combining behavioral assays, imaging and biochemical techniques, we are able to monitor protein misfolding at the resolution of the organism, the cell, and the protein, respectively.

Using Caenorhabditis elegans as a Model System to Study Protein Homeostasis in a Multicellular Organism

To study the relationship between protein homeostasis, stress and aging, we monitored changes in protein folding by following protein dysfunction, protein localization in the cell and protein stability at the organismal, cellular and protein levels, using the genetically tractable metazoan Caenorhabditis elegans as a model system.

The folding and assembly of proteins is essential for protein function, the long-term health of the cell, and longevity of the organism. Historically, the ...

Dietary Supplementation of Polyunsaturated Fatty Acids in Caenorhabditis elegans

Fatty acids are essential for numerous cellular functions. They serve as efficient energy storage molecules, make up the hydrophobic core of membranes, and participate in various signaling pathways. Caenorhabditis elegans synthesizes all of the enzymes necessary to produce a range of omega-6 and omega-3 fatty acids. This, combined with the simple anatomy and range of available genetic tools, make it an attractive model to study fatty acid function. In order to investigate the genetic pathways that mediate the physiological effects of dietary fatty acids, we have developed a method to supplement the C. elegans diet with unsaturated fatty acids. Supplementation is an effective means to alter the fatty acid composition of worms and can also be used to rescue defects in fatty acid-deficient mutants. Our method uses nematode growth medium agar (NGM) supplemented with fatty acidsodium salts. The fatty acids in the supplemented plates become incorporated into the membranes of the bacterial food source, which is then taken up by the C. elegans that feed on the supplemented bacteria. We also describe a gas chromatography protocol to monitor the changes in fatty acid composition that occur in supplemented worms. This is an efficient way to supplement the diets of both large and small populations of C. elegans, allowing for a range of applications for this method.

Dietary Supplementation of Polyunsaturated Fatty Acids in Caenorhabditis elegans

Caenorhabditis elegans is a useful model to explore the functions of polyunsaturated fatty acids in development and physiology. This protocol describes an efficient method of supplementing the C. elegans diet with polyunsaturated fatty acids.

Fatty acids are essential for numerous cellular functions. They serve as efficient energy storage molecules, make up the hydrophobic core of membranes, ...

Modeling Age-Associated Neurodegenerative Diseases in Caenorhabditis elegans

Battling human neurodegenerative pathologies and managing their pervasive socioeconomic impact is becoming a global priority. Notwithstanding their detrimental effects on the human life quality and the healthcare system, the majority of human neurodegenerative disorders still remain incurable and non-preventable. Therefore, the development of novel therapeutic interventions against such maladies is becoming a pressing urgency. Age-associated deterioration of neuronal circuits and function is evolutionarily conserved in organisms as diverse as the lowly worm Caenorhabditis elegans and humans, signifying similarities in the underlying cellular and molecular mechanisms. C. elegans is a highly malleable genetic model, which offers a well-characterized nervous system, body transparency and a diverse repertoire of genetic and imaging techniques to assess neuronal activity and quality control during ageing. Here, we introduce and describe methodologies utilizing some versatile nematode models, including hyperactivated ion channel-induced necrosis (e.g., deg-3(d) and mec-4(d)) and protein aggregate (e.g., &#945;-syunclein and poly-glutamate)-induced neurotoxicity, to monitor and dissect the cellular and molecular underpinnings of age-related neuronal breakdown. A combination of these animal neurodegeneration models, together with genetic and pharmacological screens for cell death modulators will lead to an unprecedented understanding of age-related breakdown of neuronal function and will provide critical insights with broad relevance to human health and quality of life.

Modeling Age-Associated Neurodegenerative Diseases in Caenorhabditis elegans

Here, we introduce and describe widely accessible methodologies utilizing some versatile nematode models, including hyperactivated ion channel-induced necrosis and protein aggregate-induced neurotoxicity, to monitor and dissect the cellular and molecular underpinnings of age-associated neurodegenerative diseases.

Battling human neurodegenerative pathologies and managing their pervasive socioeconomic impact is becoming a global priority. Notwithstanding their ...

Assessment of de novo Protein Synthesis Rates in Caenorhabditis elegans

Maintaining a healthy proteome is essential for cell and organismal homeostasis. Perturbation of the balance between protein translational control and degradation instigates a multitude of age-related diseases. Decline of proteostasis quality control mechanisms is a hallmark of ageing. Biochemical methods to detect de novo protein synthesis are still limited, have several disadvantages and cannot be performed in live cells or animals. Caenorhabditis elegans, being transparent and easily genetically modified, is an excellent model to monitor protein synthesis rates by using imaging techniques. Here, we introduce and describe a method to measure de novo protein synthesis in vivo utilizing fluorescence recovery after photobleaching (FRAP). Transgenic animals expressing fluorescent proteins in specific cells or tissues are irradiated by a powerful light source resulting in fluorescence photobleaching. In turn, assessment of fluorescence recovery signifies new protein synthesis in cells and/or tissues of interest. Hence, the combination of transgenic nematodes, genetic and/or pharmacological interventions together with live imaging of protein synthesis rates can shed light on mechanisms mediating age-dependent proteostasis collapse.

Assessment of de novo Protein Synthesis Rates in Caenorhabditis elegans

Here, we introduce and describe a nonradioactive and noninvasive method to assess de novo protein synthesis in vivo, utilizing the nematode Caenorhabditis elegans and fluorescence recovery after photobleaching (FRAP). This method can be combined with genetic and/or pharmacological screens to identify novel modulators of protein synthesis.

Maintaining a healthy proteome is essential for cell and organismal homeostasis. Perturbation of the balance between protein translational control and ...

Lipid Supplementation for Longevity and Gene Transcriptional Analysis in Caenorhabditis elegans

Aging is a complex process characterized by progressive physiological changes resulting from both environmental and genetic contributions. Lipids are crucial in constituting structural components of cell membranes, storing energy, and as signaling molecules. Regulation of lipid metabolism and signaling is essential to activate distinct longevity pathways. The roundworm Caenorhabditis elegans is an excellent and powerful organism to dissect the contribution of lipid metabolism and signaling in longevity regulation. Multiple research studies have described how diet supplementation of specific lipid molecules can extend C. elegans lifespan; however, minor differences in the supplementation conditions can cause reproducibility issues among scientists in different labs. Here, two detailed supplementation methods for C. elegans are reported employing lipid supplementation either with bacteria seeded on plates or bacterial suspension in liquid culture. Also provided herein are the details to perform lifespan assays with lifelong lipid supplementation and qRT-PCR analysis using a whole worm lysate or dissected tissues derived from a few worms. Using a combination of longitudinal studies and transcriptional investigations upon lipid supplementation, the feeding assays provide dependable approaches to dissect how lipids influence longevity and healthy aging. This methodology can also be adapted for various nutritional screening approaches to assess changes in a subset of transcripts using either a small number of dissected tissues or a few animals.

Lipid Supplementation for Longevity and Gene Transcriptional Analysis in Caenorhabditis elegans

The present protocol describes lipid supplementation methods in liquid and on-plate cultures for Caenorhabditis elegans, coupled with longitudinal studies and gene transcriptional analysis from bulk or a few worms and worm tissues.

Aging is a complex process characterized by progressive physiological changes resulting from both environmental and genetic contributions. Lipids are ...

Application of Flow Vermimetry for Quantification and Analysis of the Caenorhabditis elegans Gut Microbiome

The composition of the gut microbiome can have a dramatic impact on host physiology throughout the development and the life of the animal. Measuring compositional changes in the microbiome is crucial in identifying the functional relationships between these physiological changes. Caenorhabditis elegans has emerged as a powerful host system to examine the molecular drivers of host-microbiome interactions. With its transparent body plan and fluorescent-tagged natural microbes, the relative levels of microbes within the gut microbiome of an individual C. elegans animal can be easily quantified using a large particle sorter. Here we describe the procedures for the experimental setup of a microbiome, collection, and analysis of C. elegans populations in the desired life stage, operation, and maintenance of the sorter, and statistical analyses of the resulting datasets. We also discuss considerations for optimizing sorter settings based on the microbes of interest, the development of effective gating strategies for C. elegans life stages, and how to utilize sorter capabilities to enrich animal populations based on gut microbiome composition. Examples of potential applications will be presented as part of the protocol, including the potential for scalability to high-throughput applications.

Application of Flow Vermimetry for Quantification and Analysis of the Caenorhabditis elegans Gut Microbiome

Caenorhabditis elegans is a powerful model to examine the molecular determinants driving host-microbiome interactions. We present a high throughput pipeline profiling the single animal levels of gut microbiome colonization together with key aspects of the C. elegans physiology.

The composition of the gut microbiome can have a dramatic impact on host physiology throughout the development and the life of the animal. Measuring ...

Quantifying Mitophagy in C. elegans and a Liver Cancer Cell Line

Detection of Mitophagy in Caenorhabditis elegans and Mammalian Cells Using Organelle-Specific Dyes

Mitochondria are essential for various biological functions, including energy production, lipid metabolism, calcium homeostasis, heme biosynthesis, regulated cell death, and the generation of reactive oxygen species (ROS). ROS are vital for key biological processes. However, when uncontrolled, they can lead to oxidative injury, including mitochondrial damage. Damaged mitochondria release more ROS, thereby intensifying cellular injury and the disease state. A homeostatic process named mitochondrial autophagy (mitophagy) selectively removes damaged mitochondria, which are then replaced by new ones. There are multiple mitophagy pathways, with the common endpoint being the breakdown of the damaged mitochondria in lysosomes. 
Several methodologies, including genetic sensors, antibody immunofluorescence, and electron microscopy, use this endpoint to quantify mitophagy. Each method for examining mitophagy has its advantages, such as specific tissue/cell targeting (with genetic sensors) and great detail (with electron microscopy). However, these methods often require expensive resources, trained personnel, and a lengthy preparation time before the actual experiment, such as for creating transgenic animals. Here, we present a cost-effective alternative for measuring mitophagy using commercially available fluorescent dyes targeting mitochondria and lysosomes. This method effectively measures mitophagy in the nematode Caenorhabditis elegans and human liver cells, which indicates its potential efficiency in other model systems.

Author Spotlight: Detection of Mitophagy in Caenorhabditis elegans and Mammalian Cells Using Organelle-Specific Dyes  

Exploring mitophagy through electron microscopy, genetic sensors, and immunofluorescence requires costly equipment, skilled personnel, and a significant time investment. Here, we demonstrate the efficacy of a commercial fluorescence dye kit in quantifying the mitophagy process in both Caenorhabditis elegans and a liver cancer cell line.

Mitochondria are essential for various biological functions, including energy production, lipid metabolism, calcium homeostasis, heme biosynthesis, ...