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

Summary

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.

Abstract

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.

Introduction

Animal evolution is under constant microbial influence¹. From diverse microbes in the environment, animal hosts acquire specific partners² that extend the capabilities of the host and drive its physiology and susceptibility to disease³. For example, metagenomic analyses of the gut microbiome uncovered enriched metabolic classes of microbial genes that may confer greater energy harvest and storage in obese mice⁴, many of which are also found in the human gut microbiome⁵. There is still a great need to establish causal relationshi....

Protocol

1. Preparation of microbiome mixture

1. Stamp or streak out bacteria from a glycerol freezer stock onto a lysogeny broth (LB) plate, or appropriate growth medium, and grow overnight at an optimal temperature based on bacterial strains of interest (typically 25 °C for C. elegans natural microbes).
2. From the LB plate, use a single colony from each bacterial isolate (e.g., 12 bacteria of the CeMbio collection) to inoculate 800 µL of LB medium in separate wells of a 1 mL deep-well plate. Incubate overnight at the optimal temperature with shaking at 250 rpm.
   NOTE: This protocol is optimized for use with defined n....

Results

Defining adult and larvae population gates
Here, synchronized C. elegans L1s were grown on an NGM plate seeded with E. coli OP50 (Eco), a standard laboratory diet. C. elegans populations were collected for LPS analysis after 96 h or 120 h of growth at 20 °C (Figure 2A). A dot plot of extinction (EXT, a proxy of body density) versus time-of-flight (TOF, a proxy of body length) creates two visually separated clouds of animals. Each dot represents.......

Discussion

Flow vermimetry has been used to characterize C. elegans genes and pathways against pathogen colonization and toxicity in several studies^21,22. Here, a high throughput amenable approach is presented that uses C. elegans to investigate how intestinal microbiomes modulate their host physiology. Compared to existing methods using colony forming units (CFU) or 16S rRNA amplicon sequencing^9,16

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
15 mL conical bottom centrifuge tubes	VWR	10026-076
96 deep-well plates (1 mL)	Axygen	P-DW-11-C
96 deep-well plates (2 mL)	Axygen	P-DW-20-C
96-well Costar plate	Corning	3694
Agar	Millipore Sigma		Standard bacteriology agar is also sufficient.
Aspirating manifold	V&P scientific	VP1171A
Bleach	Clorox
Bleach solution			Mix Bleach with 5M Sodium hypochlorite 2:1 (v/v)
Cell Imaging Multimode Reader	Biotek	Cytation 5	Bacterial OD measurement
Centrifuge	Thermo scientific	Sorvall Legend XTR	For 96 well plate and conical tubes
Fluorescent Microscope	Nikon	TiE
ggplot: Various R Programming Tools for Plotting Data.	R package	Version 3.3.2
Large Particle Autosampler	Union Biometrica	LP Sampler
Large Particle Sorter	Union Biometrica	COPAS Biosorter
Levamisole	Fisher	AC187870100
Lysogeny Broth (LB)	RPI	L24066	Standard LB home-made recipes using Bacto-tryptone, yeast extract, and NaCl are also sufficient.
M9 solution			22 mM KH2PO4 monobasic, 42.3 mM Na2HPO4, 85.6 mM NaCl, 1 mM MgSO4
Nematode Growth Medium	RPI	N81800-1000.0	1 mM CaCl2, 25 mM KPO4 pH 6.0, 1 mM MgSO4 added after autoclaving.
RStudio	GNU	Version 1.3.1093
Sodium hypochlorite	Sigma-Aldrich		5M NaOH
Stereo Microscope	Nikon	SMZ745
Sterile 10 cm diameter petri dishes	Corning	351029
Sterile 12-well plates	VWR	10062-894
Sterile 24-well plates	VWR	10062-896
Sterile 6 cm diameter petri dishes	Corning	351007
Triton X-100	Sigma-Aldrich	T8787