Selective Cleaning of Wild <em>Caenorhabditis</em> Nematodes to Enrich for Intestinal Microbiome Bacteria

Abstract

Caenorhabditis elegans (C. elegans) 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. F 1 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.

RNA fluorescence in situ hybridization (FISH) to visualize microbial colonization and infection in the Caenorhabditis elegans intestines

“…2A). JU1848 was then selectively cleaned to eliminate other microbial contaminants and enrich for the desired adhering bacterium 21 . The bacterium was identified as a new species in the Enterobacteriaceae family.…”

“…Exp . (174), e62937, doi:10.3791/62937 (2021) 21 . (B) FISH staining of JU1848 using a red labeled probe designed to the 16S rRNA sequence of the adhering bacterium (top) and a green labeled universal FISH probe to the 16S of bacteria (bottom).…”

“…Importantly, Orsay virus and microsporidia are obligate parasites and cannot be cultured independently of the host, and there are no genetically modified versions of pathogens that express a fluorescent protein, so FISH is the standard visualization tool. In addition to staining microorganisms in C. elegans intestine, this protocol can be used for other nematode strains like C. tropicalis or Oscheius tipulae 17,21 .…”

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

“…Under the differential interference contrast (DIC) microscope, this nematode strain was found to be colonized with a bacterium that appears to directionally adhere to the intestinal epithelium (Figure 2A). JU1848 was then selectively cleaned to eliminate other microbial contaminants and enrich for the desired adhering bacterium 23 . Using the universal PCR method, the bacterium was identified as a new species in The bipartite RNA genome of the Orsay virus consists of RNA1 and RNA2 segments, and FISH probes targeting both of these segments have been developed (Figure 3A,B) 9 , 18 .…”

“…Colonization or infection can also be quantified through RNA FISH using nematodes grown on plates seeded with a desired culturable bacteria of interest. In addition to staining microorganisms in C. elegans intestine, this protocol can be used for other nematode strains like C. tropicalis or Oscheius tipulae 19 , 23 .…”