The authors would like to acknowledge H. Schulenberg and C. LaRock for their generous sharing of bacterial strains used in these experiments. This work was supported by funding from Emory University and NSF (PHY2014173).

<ol>
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The authors have no conflicts of interest.

Here data are presented on the advantages of single-worm quantification of bacterial load in C. elegans, along with a 96-well disruption protocol to allow the rapid and consistent acquisition of large data sets of this type. As compared with existing methods33, these protocols allow higher-throughput measurement of intestinal microbial communities in the worm.
This approach has plating as a rate-limiting step and is not truly &#34;high-throughput&#34;. Large-object flow cytometry (Figure 3C,D) is a useful high-throughput method for quantifying fluorescently labeled bacteria in individual worms16, although the number of simultaneous fluorophores is a limitation in multi-species communities. Linking multi-well plate disruption with community sequencing is another way to increase throughput; however, the 96-well disruption procedure described here was optimized specifically to leave bacterial cells intact. Sequencing-based analysis, where thorough lysis of cells is desirable, will require addition of a nucleic acid extraction step or modification of the beating protocol (Protocol 3.10-3.11) to extract cell contents instead of live bacteria. Protocols for single-worm disruption and extraction of nucleic acids have been published elsewhere38,39.
Bacterial total abundance in the worm intestine is heterogeneous, and the data shown here suggest that batch-based measurement can produce erroneous results in comparisons between groups. However, other measures of bacterial communities in the worm may be less sensitive to the effects of batching. Of note, relative abundances in worm-associated communities seem to vary very little if at all with total intestinal population size, regardless of whether interactions among microbes are neutral40 or not14. It is plausible that, compared to count data, relative abundance measures will be less susceptible to the false-positive rate issue described. Sequencing-based community analysis, which generates relative abundance data for community composition, may therefore not require measurement of single worms. Further investigation is needed on this point.
Here, we use cold treatment to paralyze worms for surface bleaching. Other work has found that worms resume normal activity rapidly (&#60;15 min) if time on ice is kept under 30 min, allowing immediate use in further assays, in contrast with chemical paralysis agents which can require extended periods before full recovery34. If worms are to be disrupted immediately for bacterial quantification, this feature is dispensable, and the main advantage of chilling vs. chemical paralysis is avoiding the need for a controlled waste stream. Extended cold treatment should be used with caution when investigating stress responses, particularly if there is a known connection to temperature. The cold paralysis protocols described here entail shorter acute cold exposure than used in experiments for cold stress (20-30 min vs 2+ h at 2-4&#176;C)41,42,43, and a 1 h cold shock produces no apparent phenotype in wild-type worms43. Short-term (90 min) incubation at 4 &#176;C induces changes in cold-stress gene expression (measured by expression of a TMEM-135::GFP reporter), but expression returns to unstressed levels within minutes once worms are returned to room temperature34. However, the effects on stress-sensitive worm genotypes may be more severe than in wild-type. This procedure should be validated under the experimental conditions to be used.
The surface bleaching protocol described here can be used as a way to limit or eliminate passaging of external microbes in experiments. This method has additionally been used to clear fungal contaminants by surface bleaching and transferring only L1/L2 larvae to fresh plates (transfer of surface-bleached adults resulted in failure to clear the contaminant, presumably due to carriage in the intestines of the larger animals). It is critically important to ensure that bleach concentration does not exceed 1:1000 v/v, as damage to the worms and mortality will result. This procedure may be useful in experimental host-microbe evolution and host-pathogen interactions. For example, the low excretion rate of live bacteria observed here can help to explain the highly variable rates observed for bacterial transmission from hermaphrodites to offspring15. The lack of correlation between intestinal bacterial load and excretion rate observed here is interesting but requires further investigation; a larger number of data points across a range of conditions will be needed to determine where (or whether) this observation will hold.
It may not always be necessary to clean worms to the extent provided by surface bleaching. Multiple washes in sterile buffer are likely sufficient when worms are internally colonized with a single microbe if the minimum expected CFU/worm is much higher (10-100-fold) than the concentration of bacteria in buffer supernatant, as this carryover will minimally affect counts (see Figure 1). Additionally, if the microbe(s) of interest primarily colonize(s) the cuticle, surface bleaching should clearly be avoided. Thorough cleaning is more important to ensure accuracy when dealing with mixed microbial communities (to ensure that all colonies/reads in a sample are from worm-associated bacteria and not from the environment), when bacteria adhered to the cuticle interfere with reading the internalized population, when expected minimum CFU/worm is low, etc.

Heterogeneity in host-microbe associations is observed ubiquitously, and variation between individuals is increasingly recognized as a contributing factor in population-level processes from competition and coexistence1 to disease transmission2,3,4. In C. elegans, &#34;hidden heterogeneity&#34; within isogenic populations has been observed repeatedly, with sub-populations of individuals showing distinct phenotypes in heat shock response5,6, ageing, and lifespan7,8,9,10,11, and many other aspects of physiology and development12. Most analyses that attempt to identify sub-population structure provide evidence for two sub-populations in experimental populations of isogenic, synchronized worms5,7,8, though other data suggest the possibility of within-population distributions of traits rather than distinct groups7,12,13. Of relevance here, substantial heterogeneity in intestinal populations is observed even within isogenic populations of worms colonized from a shared source of microbes13,14,15,16, and this heterogeneity can be concealed by the batch digest measurements that are widely used17,18,19,20 for bacterial quantification in the worm.
This work presents data suggesting a need for greater reliance on single-worm measurements in host-microbe association, as well as protocols for increasing accuracy and throughput in single-worm disruption. These protocols are designed to facilitate mechanical disruption of large numbers of individual C. elegans for quantification of viable bacterial load, while providing better repeatability and lower effort per sample than pestle-based disruption of individual worms. A recommended gut-purging step, where worms are permitted to feed on heat-killed E. coli prior to the preparation for disruption, is included to minimize contributions from recently ingested and other transient (non-adhered) bacteria. These protocols include a cold-paralysis method for cleaning the cuticle with a low-concentration surface bleach treatment; surface bleaching can be used as a preparatory step in single-worm disruption or as a method for preparing live, externally germ-free worms. This surface-bleaching method is sufficient to remove a wide range of external microbes, and cold treatment provides an alternative to conventional levamisole-based paralysis; while levamisole will be preferred for cold-sensitive experiments, cold paralysis minimizes contributions to hazardous waste streams and allows rapid resumption of normal activity. While the full protocol describes a laboratory experiment where worms are colonized with known bacteria, the procedures for cleaning worms and single-worm disruption can readily be applied to worms isolated from wild samples or colonized in microcosm experiments. The protocols described here produces live bacteria extracted from the worm intestine, suitable for plating and quantification of colony forming units (CFUs) in individual worms; for sequencing-based intestinal community analysis, subsequent cell lysis and nucleic acid extraction steps should be added to these protocols.

<table><tbody><tr><td>96-well flat-bottom polypropylene plates, 300 uL</td><td>Evergreen Labware</td><td>290-8350-03F</td><td></td></tr><tr><td>96-well plate sealing mat, silicon, square wells (AxyMat)</td><td>Axygen</td><td>AM-2ML-SQ</td><td></td></tr><tr><td>96-well plates, 2 mL, square wells</td><td>Axygen</td><td>P-2ML-SQ-C-S</td><td></td></tr><tr><td>96-well polypropylene plate lids</td><td>Evergreen Labware</td><td>290-8020-03L</td><td></td></tr><tr><td>Agar</td><td>Fisher Scientific</td><td>443570050</td><td></td></tr><tr><td>Bead mill adapter set for 96-well plates</td><td>QIAGEN</td><td>119900</td><td>Adapter plates for use with two 96-well plates on the TissueLyser II</td></tr><tr><td>Bead mill tissue homogenizer (TissueLyser II)</td><td>QIAGEN</td><td>85300</td><td>Mechanical homogenizer for medium to high-throughput sample disruption</td></tr><tr><td>BioSorter</td><td>Union Biometrica</td><td>By quotation</td><td>Large object sorter equipped with a 250 micron focus for &lt;em&gt;C. elegans&lt;/em&gt;</td></tr><tr><td>Bleach, commercial, 8.25% sodium hypochlorite</td><td>Clorox</td><td></td><td></td></tr><tr><td>Breathe-Easy 96-well gas permeable sealing membrane</td><td>Diversified Biotech</td><td>BEM-1</td><td>Multiwell plate gas permeable polyurethane membranes. Thin sealing film is permeable to O2, CO2, and water vapors and is UV transparent down to 300 nm. Sterile, 100/box.</td></tr><tr><td>Calcium chloride dihydrate</td><td>Fisher Scientific</td><td>AC423525000</td><td></td></tr><tr><td>Cholesterol</td><td>VWR</td><td>AAA11470-30</td><td></td></tr><tr><td>Citric acid monohydrate</td><td>Fisher Scientific</td><td>AC124910010</td><td></td></tr><tr><td>Copper (II) sulfate pentahydrate</td><td>Fisher Scientific</td><td>AC197722500</td><td></td></tr><tr><td>Corning 6765 LSE Mini Microcentrifuge</td><td>Corning</td><td>&amp;nbsp;COR-6765</td><td></td></tr><tr><td>Disodium EDTA</td><td>Fisher Scientific</td><td>409971000</td><td></td></tr><tr><td>DL 1,4 Dithiothreitol, 99+%, for mol biology, DNAse, RNAse and Protease free, ACROS Organics</td><td>Fisher Scientific</td><td>327190010</td><td></td></tr><tr><td>Eppendorf 1.5 mL microcentrifuge tubes, natural</td><td>Eppendorf</td><td></td><td></td></tr><tr><td>Eppendorf 5424R microcentrifuge</td><td>Eppendorf</td><td>5406000640</td><td>24-place refrigerated benchtop microcentrifuge</td></tr><tr><td>Eppendorf 5810R centrifuge with rotor S-4-104</td><td>Eppendorf</td><td>22627040</td><td>3L benchtop centrifuge with adaptors for 15-50 mL tubes and plates</td></tr><tr><td>Eppendorf plate bucket (x2), for Rotor S-4-104</td><td>Eppendorf</td><td>22638930</td><td></td></tr><tr><td>Ethanol 100%</td><td>Fisher Scientific</td><td>BP2818500</td><td></td></tr><tr><td>Glass beads, 2.7 mm</td><td>Life Science Products</td><td>LS-79127</td><td></td></tr><tr><td>Glass beads, acid-washed, 425-600 &amp;micro;m</td><td>Sigma</td><td>G877-500G</td><td></td></tr><tr><td>Glass plating beads</td><td>VWR</td><td>76005-124</td><td></td></tr><tr><td>Hydrochloric acid</td><td>VWR</td><td>BDH7204-1</td><td></td></tr><tr><td>Iron (II) sulfate heptahydrate</td><td>Fisher Scientific</td><td>423731000</td><td></td></tr><tr><td>Kimble Kontes pellet pestle motor</td><td>DWK Life Sciences</td><td>749540-0000</td><td></td></tr><tr><td>Kimble Kontes polypropylene pellet pestles and microtubes, 0.5 mL</td><td>DWK Life Sciences</td><td>749520-0590</td><td></td></tr><tr><td>Leica DMi8 motorized inverted microscope with motorized stage</td><td>Leica</td><td>11889113</td><td></td></tr><tr><td>Leica LAS X Premium software</td><td>Leica</td><td>11640687</td><td></td></tr><tr><td>Magnesium sulfate heptahydrate</td><td>Fisher Scientific</td><td>AC124900010</td><td></td></tr><tr><td>Manganese(II) chloride tetrahydrate</td><td>VWR</td><td>470301-706</td><td></td></tr><tr><td>PARAFILM M flexible laboratory sealing film</td><td>Amcor</td><td>PM996</td><td></td></tr><tr><td>Peptone</td><td>Fisher Scientific</td><td>BP1420-500</td><td></td></tr><tr><td>Petri dishes, round, 10 cm</td><td>VWR</td><td>25384-094</td><td></td></tr><tr><td>Petri dishes, round, 6 cm</td><td>VWR</td><td>25384-092</td><td></td></tr><tr><td>Petri dishes, square, 10 x 10 cm</td><td>VWR</td><td>10799-140</td><td></td></tr><tr><td>Phospho-buffered saline (1X PBS)</td><td>Gold Bio</td><td>P-271-200</td><td></td></tr><tr><td>Polypropylene autoclave tray, shallow</td><td>Fisher Scientific</td><td>13-361-10</td><td></td></tr><tr><td>Potassium hydroxide</td><td>Fisher Scientific</td><td>AC134062500</td><td></td></tr><tr><td>Potassium phosphate dibasic</td><td>Fisher Scientific</td><td>BP363-1</td><td></td></tr><tr><td>Potassium phosphate monobasic</td><td>Fisher Scientific</td><td>BP362-1</td><td></td></tr><tr><td>R 4.1.3/RStudio 2022.02.0 build 443</td><td>R Foundation</td><td>n/a</td><td></td></tr><tr><td>Scoop-type laboratory spatula, metal</td><td>VWR</td><td>470149-438</td><td></td></tr><tr><td>Silicon carbide 36 grit</td><td>MJR Tumblers</td><td>n/a</td><td>Black extra coarse silicon carbide grit. Available in 0.5-5 lb sizes from this vendor.</td></tr><tr><td>Sodium dodecyl sulfate</td><td>Fisher Scientific</td><td>BP166-100</td><td></td></tr><tr><td>Sodium hydroxide</td><td>VWR</td><td>BDH7247-1</td><td></td></tr><tr><td>Sodium phosphate dibasic anhydrous</td><td>Fisher Scientific</td><td>BP332-500</td><td></td></tr><tr><td>Sodum chloride</td><td>Fisher Scientific</td><td>BP358-1</td><td></td></tr><tr><td>Sucrose</td><td>Fisher Scientific</td><td>AC419760010</td><td></td></tr><tr><td>Tri-potassium citrate monohydrate</td><td>Fisher Scientific</td><td>AC611755000</td><td></td></tr><tr><td>Triton X-100</td><td>Fisher Scientific</td><td>BP151-100</td><td></td></tr><tr><td>Zinc sulfate heptahydrate</td><td>Fisher Scientific</td><td>AC205982500</td><td></td></tr></tbody></table>

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.

Watch this Scientific Journal Video about Using Single-Worm Data to Quantify Heterogeneity in Caenorhabditis elegans-Bacterial Interactions at JoVE.com

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.

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 ...

using-single-worm-data-to-quantify-heterogeneity-caenorhabditis

Nanyang Technological University

Research

JoVE Journal

Biology

2.0K Views.  Emory University. This protocol describes a 96-well disruption of individual bacterially colonized Caenorhabditis elegans following cold paralysis and surface bleaching 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.

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

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

High-Throughput Quantitative RT-PCR in Single and Bulk C. elegans Samples Using Nanofluidic Technology

This paper presents a high-throughput reverse transcription quantitative PCR (RT-qPCR) assay for Caenorhabditis elegans that is fast, robust, and highly sensitive. This protocol obtains precise measurements of gene expression from single worms or from bulk samples. The protocol presented here provides a novel adaptation of existing methods for complementary DNA (cDNA) preparation coupled to a nanofluidic RT-qPCR platform. The first part of this protocol, named &#8216;Worm-to-CT&#8217;, allows cDNA production directly from nematodes without the need for prior mRNA isolation. It increases experimental throughput by allowing the preparation of cDNA from 96 worms in 3.5 h. The second part of the protocol uses existing nanofluidic technology to run high-throughput RT-qPCR on the cDNA. This paper evaluates two different nanofluidic chips: the first runs 96 samples and 96 targets, resulting in 9,216 reactions in approximately 1.5 days of benchwork. The second chip type consists of six 12 x 12 arrays, resulting in 864 reactions. Here, the Worm-to-CT method is demonstrated by quantifying mRNA levels of genes encoding heat shock proteins from single worms and from bulk samples. Provided is an extensive list of primers designed to amplify processed RNA for the majority of coding genes within the C. elegans genome.

High-Throughput Quantitative RT-PCR in Single and Bulk C. elegans Samples Using Nanofluidic Technology

In this article a high-throughput protocol for fast and reliable determination of gene expression levels in single or bulk C. elegans samples is described. This protocol does not require RNA isolation and produces cDNA directly from samples. It can be used together with high-throughput multiplexed nanofluidic real-time qPCR platforms.

This paper presents a high-throughput reverse transcription quantitative PCR (RT-qPCR) assay for Caenorhabditis elegans that is fast, robust, and highly ...

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 ...

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

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

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

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

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

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

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

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

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

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

Immunology and Infection

High-Throughput Assay to Measure Caenorhabditis elegans Food Intake and Lifespan

Quantifying Food Intake in Caenorhabditis elegans by Measuring Bacterial Clearance

Feeding is an essential biological process for an organism&#39;s growth, reproduction, and survival. This assay aims to measure the food intake of Caenorhabditis elegans&#160;(C. elegans), an important parameter when studying the genetics of aging or metabolism. In most species, feeding is determined by measuring the difference between the amount of food provided and the amount left after a given time interval. The method presented here uses the same strategy to determine the feeding of C. elegans. It measures the amount of bacteria, the food source of C. elegans, cleared within 72 h. This method uses 96-well microtiter plates and has allowed the screening of hundreds of drugs for their ability to modulate food intake at a speed and depth not possible in other animal models. The strength of this assay is that it allows to measure feeding and lifespan simultaneously and directly measures the disappearance of food and, thus, is based on the same principles used for other organisms, facilitating species-to-species comparison.

Author Spotlight: Exploring the Relationship Between Lipotoxicity and HFpEF 

This protocol describes an assay to quantify Caenorhabditis elegans feeding rate based on measuring the clearance of bacteria in liquid culture.

Feeding is an essential biological process for an organism&#39;s growth, reproduction, and survival. This assay aims to measure the food intake of ...

Behavior

Automating Aggregate Quantification in Caenorhabditis elegans

A rise in the prevalence of neurodegenerative protein conformational diseases (PCDs) has fostered a great interest in this subject over the years. This increased attention has called for the diversification and improvement of animal models capable of reproducing disease phenotypes observed in humans with PCDs. Though murine models have proven invaluable, they are expensive and are associated with laborious, low-throughput methods. Use of the Caenorhabditis elegans nematode model to study PCDs has been justified by its relative ease of maintenance, low cost, and rapid generation time, which allow for high-throughput applications. Additionally, high conservation between the C. elegans and human genomes makes this model an invaluable discovery tool. Nematodes that express fluorescently tagged tissue-specific polyglutamine (polyQ) tracts exhibit age- and polyQ length-dependent aggregation characterized by fluorescent foci. Such reporters are often employed as proxies to monitor changes in proteostasis across tissues. Manual aggregate quantification is time-consuming, limiting experimental throughput. Furthermore, manual foci quantification can introduce bias, as aggregate identification can be highly subjective. Herein, a protocol consisting of worm culturing, image acquisition, and data processing was standardized to support high-throughput aggregate quantification using C. elegans that express intestine-specific polyQ. By implementing a C. elegans-based image processing pipeline using CellProfiler, an image analysis software, this method has been optimized to separate and identify individual worms and enumerate their respective aggregates. Though the concept of automation is not entirely unique, the need to standardize such procedures for reproducibility, elimination of bias from manual counting, and increase throughput is high. It is anticipated that these methods can drastically simplify the screening process of large bacterial, genomic, or drug libraries using the C. elegans model.

Automating Aggregate Quantification in Caenorhabditis elegans

The following protocol describes the development and optimization of a high-throughput workflow for worm culturing, fluorescence imaging, and automated image processing to quantify polyglutamine aggregates as an assessment of changes in proteostasis.

A rise in the prevalence of neurodegenerative protein conformational diseases (PCDs) has fostered a great interest in this subject over the years. This ...

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 ...

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 ...

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.

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.

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

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

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

High-Throughput Quantitative RT-PCR in Single and Bulk C. elegans Samples Using Nanofluidic Technology

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

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

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

Quantifying Food Intake in Caenorhabditis elegans by Measuring Bacterial Clearance

Automating Aggregate Quantification in Caenorhabditis elegans

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

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

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