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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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><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>KH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;</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>MgSO&lt;sub&gt;4&lt;/sub&gt;</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>Na&lt;sub&gt;2&lt;/sub&gt;HPO&lt;sub&gt;4&lt;/sub&gt;</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 &amp;amp; 1.6</td></tr><tr><td>Qubit Fluorometer</td><td>Invitrogen</td><td>Q33238</td><td>Supplementary Steps 1.1 &amp;amp; 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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Biology

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

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

Culture and Assay of Large-Scale Mixed-Stage Caenorhabditis elegans Populations

Caenorhabditis elegans (C. elegans) has been and remains a valuable model organism to study developmental biology, aging, neurobiology, and genetics. The large body of work on C. elegans makes it an ideal candidate to integrate into large-population, whole-animal studies to dissect the complex biological components and their relationships with another organism. In order to use C. elegans in collaborative -omics research, a method is needed to generate large populations of animals where a single sample can be split and assayed across diverse platforms for comparative analyses.
Here, a method to culture and collect an abundant mixed-stage C. elegans population on a large-scale culture plate (LSCP) and subsequent phenotypic data is presented. This pipeline yields sufficient numbers of animals to collect phenotypic and population data, along with any data needed for -omics experiments (i.e., genomics, transcriptomics, proteomics, and metabolomics). In addition, the LSCP method requires minimal manipulation to the animals themselves, less user preparation time, provides tight environmental control, and ensures that handling of each sample is consistent throughout the study for overall reproducibility. Lastly, methods to document population size and population distribution of C. elegans life stages in a given LSCP are presented.

Culture and Assay of Large-Scale Mixed-Stage Caenorhabditis elegans Populations

To use Caenorhabditis elegans (C. elegans) in omics research, a method is needed to generate large populations of worms where a single sample can be measured across platforms for comparative analyses. Here, a method to culture C. elegans populations on large-scale culture plates (LSCPs) and to document population growth is presented.

Caenorhabditis elegans (C. elegans) has been and remains a valuable model organism to study developmental biology, aging, neurobiology, and genetics. The ...

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 ...

Immunology and Infection

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 ...

A Highly Scalable Approach to Perform Ecological Surveys of Selfing Caenorhabditis Nematodes

Caenorhabditis elegans is one of the major model organisms in biology, but only recently have researchers focused on its natural ecology. The relative sparsity of information about C. elegans in its natural context comes from the challenges involved in the identification of the small nematode in nature. Despite these challenges, an increasing focus on the ecology of C. elegans has caused a wealth of new information regarding its life outside of the laboratory. The intensified search for C. elegans in nature has contributed to the discovery of many new Caenorhabditis species and revealed that congeneric nematodes frequently cohabitate in the wild, where they feed on microbial blooms associated with rotting plant material. The identification of new species has also revealed that the androdioecious mating system of males and self-fertilizing hermaphrodites has evolved three times independently within Caenorhabditis. The other two selfing species, C. briggsae and C. tropicalis, share the experimental advantages of C. elegans and have enabled comparative studies into the mechanistic basis of important traits, including self-fertilization. Despite these advances, much remains to be learned about the ecology and natural diversity of these important species. For example, we still lack functional information for many of their genes, which might only be attained through an understanding of their natural ecology. To facilitate ecological research of selfing Caenorhabditis nematodes, we developed a highly scalable method to collect nematodes from the wild. Our method makes use of mobile data collection platforms, cloud-based databases, and the R software environment to enhance researchers&#39; ability to collect nematodes from the wild, record associated ecological data, and identify wild nematodes using molecular barcodes.

A Highly Scalable Approach to Perform Ecological Surveys of Selfing Caenorhabditis Nematodes

This protocol can be used to perform large-scale ecological surveys of selfing Caenorhabditis nematodes. The primary advantage of this method is the efficient organization and analysis of ecological and molecular data associated with the nematodes collected from nature.

Caenorhabditis elegans is one of the major model organisms in biology, but only recently have researchers focused on its natural ecology. The relative ...

High-Throughput Screening of Microbial Isolates with Impact on Caenorhabditis elegans Health

With its small size, short lifespan, and easy genetics, Caenorhabditis elegans offers a convenient platform to study the impact of microbial isolates on host physiology. It also fluoresces in blue when dying, providing a convenient means of pinpointing death. This property has been exploited to develop high-throughput label-free C. elegans survival assays (LFASS). These involve time-lapse fluorescence recording of worm populations set in multiwell plates, from which population median time of death can be derived. The present study adopts the LFASS approach to screen multiple microbial isolates at once for the effects on C. elegans susceptibility to severe heat and oxidative stresses. Such microbial screening pipeline, which can notably be used to prescreen probiotics, using severe stress resistance as a proxy for host health is reported here. The protocol describes how to grow both C. elegans gut microbiota isolate collections and synchronous worm populations in multiwell arrays before combining them for the assays. The example provided covers the testing of 47 bacterial isolates and one control strain on two worm strains, in two stress assays in parallel. However, the approach pipeline is readily scalable and applicable to the screening of many other modalities. Thus, it provides a versatile setup to rapidly survey a multiparametric landscape of biological and biochemical conditions that impact C. elegans health.

High-Throughput Screening of Microbial Isolates with Impact on Caenorhabditis elegans Health

Gut microbes may positively or negatively impact the health of their host via specific or conserved mechanisms. Caenorhabditis elegans is a convenient platform to screen for such microbes. The present protocol describes high-throughput screening of 48 bacterial isolates for impact on nematode stress resistance, used as a proxy for worm health.

With its small size, short lifespan, and easy genetics, Caenorhabditis elegans offers a convenient platform to study the impact of microbial isolates on ...

Using Single-Worm Data to Quantify Heterogeneity in Caenorhabditis elegans-Bacterial Interactions

The nematode Caenorhabditis elegans is a model system for host-microbe and host-microbiome interactions. Many studies to date use batch digests rather than individual worm samples to quantify bacterial load in this organism. Here it is argued that the large inter-individual variability seen in bacterial colonization of the C. elegans intestine is informative, and that batch digest methods discard information that is important for accurate comparison across conditions. As describing the variation inherent to these samples requires large numbers of individuals, a convenient 96-well plate protocol for disruption and colony plating of individual worms is established.

Using Single-Worm Data to Quantify Heterogeneity in Caenorhabditis elegans-Bacterial Interactions

This protocol describes a 96-well disruption of individual bacterially colonized Caenorhabditis elegans following cold paralysis and surface bleaching&#160;to remove external bacteria. The resulting suspension is plated on agar plates to allow accurate, medium-throughput quantification of bacterial load in large numbers of individual worms.

The nematode Caenorhabditis elegans is a model system for host-microbe and host-microbiome interactions. Many studies to date use batch digests rather ...

Isolation and Characterization of the Natural Microbiota of the Model Nematode Caenorhabditis elegans

The nematode Caenorhabditis elegans interacts with a large diversity of microorganisms in nature. In general, C. elegans is commonly found in rotten plant matter, especially rotten fruits like apples or on compost heaps. It is also associated with certain invertebrate hosts such as slugs and woodlice. These habitats are rich in microbes, which serve as food for C. elegans and which can also persistently colonize the nematode gut. To date, the exact diversity and consistency of the native C. elegans microbiota across habitats and geographic locations is not fully understood. Here, we describe a suitable approach for isolating C. elegans from nature and characterizing the microbiota of worms. Nematodes can be easily isolated from compost material, rotting apples, slugs, or attracted by placing apples on compost heaps. The prime time for finding C. elegans in the Northern Hemisphere is from September until November. Worms can be washed out of collected substrate material by immersing the substrate in buffer solution, followed by the collection of nematodes and their transfer onto nematode growth medium or PCR buffer for subsequent analysis. We further illustrate how the samples can be used to isolate and purify the worm-associated microorganisms and to process worms for 16S ribosomal RNA analysis of microbiota community composition. Overall, the described methods may stimulate new research on the characterization of the C. elegans microbiota across habitats and geographic locations, thereby helping to obtain a comprehensive understanding of the diversity and stability of the nematode's microbiota as a basis for future functional research.

Isolation and Characterization of the Natural Microbiota of the Model Nematode Caenorhabditis elegans

Caenorhabditis elegans is one of the main model species in biology, yet almost all research is performed in the absence of its naturally associated microbes. The methods described here will help to improve our understanding of the diversity of associated microbes as a basis for future functional C. elegans research.

The nematode Caenorhabditis elegans interacts with a large diversity of microorganisms in nature. In general, C. elegans is commonly found in rotten plant ...

Long-Term Culture and Monitoring of Isolated Caenorhabditis elegans on Solid Media in Multi-Well Devices

The nematode Caenorhabditis elegans is among the most common model systems used in aging research owing to its simple and inexpensive culture techniques, rapid reproduction cycle (~3 days), short lifespan (~3 weeks), and numerous available tools for genetic manipulation and molecular analysis. The most common approach for conducting aging studies in C. elegans, including survival analysis, involves culturing populations of tens to hundreds of animals together on solid nematode growth media (NGM) in Petri plates. While this approach gathers data on a population of animals, most protocols do not track individual animals over time. Presented here is an optimized protocol for the long-term culturing of individual animals on microfabricated polydimethylsiloxane (PDMS) devices called WorMotels. Each device allows up to 240 animals to be cultured in small wells containing NGM, with each well isolated by a copper sulfate-containing moat that prevents the animals from fleeing. Building on the original WorMotel description, this paper provides a detailed protocol for molding, preparing, and populating each device, with descriptions of common technical complications and advice for troubleshooting. Within this protocol are techniques for the consistent loading of small-volume NGM, the consistent drying of both the NGM and bacterial food, options for delivering pharmacological interventions, instructions for and practical limitations to reusing PDMS devices, and tips for minimizing desiccation, even in low-humidity environments. This technique allows the longitudinal monitoring of various physiological parameters, including stimulated activity, unstimulated activity, body size, movement geometry, healthspan, and survival, in an environment similar to the standard technique for group culture on solid media in Petri plates. This method is compatible with high-throughput data collection when used in conjunction with automated microscopy and analysis software. Finally, the limitations of this technique are discussed, as well as a comparison of this approach to a recently developed method that uses microtrays to culture isolated nematodes on solid media.

Long-Term Culture and Monitoring of Isolated Caenorhabditis elegans on Solid Media in Multi-Well Devices

Presented here is an optimized protocol for culturing isolated individual nematodes on solid media in microfabricated multi-well devices. This approach allows individual animals to be monitored throughout their lives for a variety of phenotypes related to aging and health, including activity, body size and shape, movement geometry, and survival.

The nematode Caenorhabditis elegans is among the most common model systems used in aging research owing to its simple and inexpensive culture techniques, ...

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 ...

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

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Chapters in this video

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

Culture and Assay of Large-Scale Mixed-Stage Caenorhabditis elegans Populations

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

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

A Highly Scalable Approach to Perform Ecological Surveys of Selfing Caenorhabditis Nematodes

High-Throughput Screening of Microbial Isolates with Impact on Caenorhabditis elegans Health

Using Single-Worm Data to Quantify Heterogeneity in Caenorhabditis elegans-Bacterial Interactions

Isolation and Characterization of the Natural Microbiota of the Model Nematode Caenorhabditis elegans

Long-Term Culture and Monitoring of Isolated Caenorhabditis elegans on Solid Media in Multi-Well Devices

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