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 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.
Caenorhabditis elegans has emerged as a powerful model system to study innate immunity and host-microbe interactions in the intestinal epithelial cells1,2. Due to having a transparent body and only 20 intestinal cells, C. elegans represents a convenient system for monitoring the processes of microbial intestinal colonization and infection in the context of an intact organism. Nematode intestinal cells share multiple morphological and functional similarities with mammalian intestinal epithelial cells, making them a tractable in vivo model for dissection of processes that govern microbiome colonization and pathogen infection3,4,5,6.
Wild C. elegans feed on a variety of microbes that colonize and infect the intestine, and sampling of these nematodes has resulted in the discovery of viruses, eukaryotes (fungi, oomycetes), and bacteria that naturally associate with this host7,8,9,10. The Orsay virus was found infecting the intestine and is currently the only known natural virus of C. elegans9. Microsporidia are fungal-related obligate intracellular pathogens that are the most commonly found infection in wild-caught Caenorhabditis, with several species having been discovered infecting C. elegans and related nematodes8,11. Many bacteria are commonly found inhabiting the intestinal lumen of wild-caught C. elegans and several species have been established as a natural model for the C. elegans microbiome (CeMbio)6,12,13,14. Discovering and characterizing microbes that naturally colonize and/or infect C. elegans is essential to understanding the genetic mechanisms that govern these host-microbe interactions, as well as visualizing novel microbial processes that only occur in the context of an intact host animal.
After sampling, wild nematodes are screened via differential interference contrast (DIC) microscopy to look for phenotypes that are indicative of infection or colonization. For example, changes in the stereotypical granulated appearance of the intestinal cells can be associated with the presence of an intracellular parasite infection8. Specifically, the loss of the gut granules and decreased cytosolic viscosity are signs of viral infection, whereas the reorganization of gut granules into 'grooves' may indicate infection with microsporidia in the genus Nematocida8,9. Because there is a wide variety of microbes present in wild C. elegans samples, it can be difficult to distinguish among microbes through DIC microscopy. Information regarding the spatial distribution of microbes within the host may also be difficult to detect due to the small size of many microbes15. Additionally, culturing any particular microbes of interest in vitro is not always possible, leading to difficulties in detection and/or quantification.
RNA fluorescence in situ hybridization (FISH) provides a method to fluorescently label microbes by utilizing fluorescent probes that bind to the RNA of the small ribosomal subunit (SSU) in fixed cells. If analysis of morphological characteristics suggests a particular class of microbe, FISH probes that target specific or broad classes of such microbes can be used. For example, EUB338 is considered a universal probe for the bacterial SSU and is commonly used to detect a wide range of bacteria16. The protocol described here uses single-stranded DNA probes that are end-labeled with a fluorophore and specifically designed to be complementary to the target SSU of the microbe of interest, although there are previously designed probes available16. The main advantage of targeting the SSU of microbes is the relatively large abundance of this RNA, which typically comprises 80%-90% of all RNA in the cell, leading to staining with a very high signal-to-noise ratio17. Probes can also be designed to target RNA to detect viruses, like the Orsay virus9,18, which are often present in very high copies in infected cells if the virus is actively replicating.
Depending on the results with known probes, it may be necessary to obtain further sequence information to design more specific probes for species confirmation in situ. A common approach is to use universal primers against conserved regions of the SSU (16S for bacteria and 18S for eukaryotes) to amplify (via PCR) regions that are more divergent8. Using this sequence information, probes with more species-specificity can be designed. These FISH probes can then enable the identification of microbes in a culture-independent manner8. Additionally, RNA FISH can give insight into unique morphological colonization and infection characteristics, including filamentation or tissue localization patterns19,20. Different colored FISH probes can be used simultaneously, which allows for visual distinction between microbes in wild nematode samples, as well as observation of microbe-microbe dynamics inside a host15,20. Furthermore, RNA FISH staining can be applied to host-pathogen interaction studies where infection and colonization of a known species can be easily quantified manually or through automated approaches to provide insights on pathogen load, for example, in comparing C. elegans mutants that have either increased or decreased resistance to infection21.
NOTE: Nematodes may be fixed with either paraformaldehyde solution (PFA) or acetone. PFA allows for better visualization of morphology than acetone and can preserve signals from transgenic green fluorescent protein (GFP), which is destroyed by acetone. However, acetone fixation is necessary to permeabilize microsporidian spores to enable labeling this life stage. Furthermore, acetone can be more convenient than PFA because it is less toxic, and samples can be stored for several days in acetone in a -20 °C freezer without the need for removing the fixative. Below are two separate protocols, using either PFA solution or acetone as a fixative. For a visualization of the protocol steps, see Figure 1.
1. FISH staining with PFA fixation
2. FISH staining with acetone fixation
For analyzing microbiome bacteria, specific and universal FISH probes to bacterial 16S were utilized on wild-isolated animals. Wild Caenorhabditis tropicalis strain (JU1848) was sampled from the Nouragues forest near a small river in the French Guiana from rotting palm tree fruits22. 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 bacterium23. Using the universal PCR method, the bacterium was identified as a new species in the Alphaproteobacteria class. A FISH probe labeled with Cal Fluor Red 610 was then designed specifically to the 16S rRNA sequence of this bacterium to allow fluorescent visualization of colonization within C. tropicalis (Figure 2B). A universal 16S rRNA FISH probe capable of binding many species of bacteria (EUB338) was labeled with 6-carboxyfluorescin (FAM) and was also added to this sample. The green and red fluorescent signals overlap completely, suggesting that most bacteria colonizing the intestines are the adhering Alphaproteobacteria bacterium. These animals were fixed in PFA before staining.
For analyzing experimental infection in the lab with intracellular pathogens of known identity, Orsay virus and microsporidian-specific FISH probes were utilized on C. elegans with a wild-type background. The Orsay virus is a positive-strand RNA virus from the Nodaviridae family, and the only natural viral pathogen found in C. elegans. 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. In the intestine, viral RNA is sensed by RIG-I homolog DRH-124, which is required for activation of the transcriptional defense program named the Intracellular Pathogen Response (IPR)25,26,27. The transcription of antiviral IPR genes is at least partially controlled by the ZIP-1 transcription factor21. Here, the expression of ZIP-1::GFP is seen localized in the intestinal nuclei of cells that show positive Orsay virus FISH staining in the cytoplasm (Figure 3A)21. Multiple animals stained with Orsay-specific FISH are shown to indicate the strength of this signal for easy quantification (Figure 3B). Animals shown in Figure 3A,B were fixed in PFA.
The microsporidian parasite named Nematocida parisii, meaning nematode-killer from Paris, is an obligate intracellular pathogen of the intestine. Several FISH probes that label 18S rRNA of N. parisii have been used, including fluorescently tagged MicroA and MicroB probes. Multiple animals stained with MicroB FISH are shown to indicate the strength of this signal for easy quantification (Figure 3C). Additionally, C. elegans is infected by other closely related microsporidia. Co-infection of N2 with N. parisii and the related N. ausubeli can be distinguished using this FISH protocol by designing species-specific FISH probes that compete against each other for binding to a divergent region on the 18S rRNA (Figure 3D)28. In this example, the N. parisii FISH probe has perfect base-pairing to the N. parisii 18S rRNA, but a 7 bp mismatch to N. ausubeli 18S rRNA. The converse is true for the N. ausubeli probe. As such, each species-specific FISH probe will outcompete for binding to the cognate species 18S over the non-cognate species. Additionally, the use of DAPI to stain nuclei allows for better localization of the infection in the context of the whole animal, especially for the intestine which has large, easily identifiable nuclei. Figure 3C,D contains animals that were fixed in PFA. Later infections with N. parisii result in the development of meronts into spores. To visualize N. parisii spores, the animals must be fixed in acetone as it penetrates the spore wall better than PFA (Figure 3E,F)8. The resulting FISH staining demonstrates the small and large rod-shaped structures, that likely correspond with N. parisii spores, which are stained with N. parisii-specific probes in red.
Figure 1: Visual representation of FISH protocol. Created with Biorender.com. Please click here to view a larger version of this figure.
Figure 2: FISH staining of wild C. tropicalis JU1848 strain colonized with adhering bacteria in the intestines. (A) Nomarski image depicting thousands of thin bacilli bacteria (bac) directionally binding to the intestine (in) of JU1848, creating a hair-like phenotype within the lumen (lu). This figure panel is adapted from Morgan, E. et al. (2021)23. (B) FISH staining of JU1848, fixed in PFA, using a red labeled probe (b002_16S_A-CF610) designed to target the 16S rRNA sequence of the adhering bacterium (top) and a green-labeled universal FISH probe (EUB338-FAM) designed to target the 16S of bacteria (bottom). DAPI staining of host nuclei is shown in blue. See Table 1 for probe sequences. Please click here to view a larger version of this figure.
Figure 3: FISH staining of C. elegans infected with intracellular pathogens. (A,B) FISH staining of C. elegans expressing ZIP-1::GFP and infected with the Orsay virus, which were fixed with PFA before staining to preserve the GFP signal. Orsay 1 Red and Orsay 2 Red probes were used for pathogen staining. (A) The composite image consists of merged red and green fluorescent channels. Nuclear ZIP-1::GFP expression is induced upon Orsay virus infection and is shown in green. Autofluorescence from the gut granules is shown in yellow and indicated with yellow arrows. Dotted lines outline the nematode body. Scale bar = 25 µm. (B) The composite image consists of merged red fluorescent and DIC channels. Scale bar = 200 µm. (C,D) FISH staining of wild-type C. elegans infected with microsporidia that were fixed in PFA. (C) FISH staining of wild-type C. elegans infected with N. parisii and fixed in PFA. MicroB-CF610 probe was used for pathogen staining. The composite image consists of merged red fluorescent and DIC channels. Scale bar = 100 µm. (D) FISH staining of wild-type C. elegans co-infected with N. parisii and N. ausubeli in the intestine. The two pathogens were co-stained using a pair of specific FISH probes that compete for binding to the same region of the 18S rRNA. N. parisii was stained using MicroF-CF610 (red) and N. ausubeli was stained using MicroSp1A-FAM (green). DAPI staining of host nuclei is seen in blue. Scale bar = 25 µm. (E) FISH staining with acetone-fixed wild-type C. elegans infected with N. parisii spores. MicroA-CF610 (red) was used for staining (red). Scale bar = 15 µm. (F) Nomarski image depicting N. parisii spores seen in (E). Scale bar = 15 µm. In (E) and (F), the small and large rod-shaped structures are labeled with small and large arrows, respectively, that correspond to N. parisii spores. See Table 1 for probe sequences. The image shown in (A) is adapted from Lažetić, V. et al. (2022)21. Images shown in (B) and (C) are adapted from Reddy, K. C. et al. (2019)26. Images shown in (E) and (F) are adapted from Troemel, E. R. et al. (2008)8. Please click here to view a larger version of this figure.
Probe name | Probe specificity | Probe fluorophore | Probe sequence |
EUB338-FAM | Bacterial 16S (universal) | 5' 6-fluorescein (FAM) | GCTGCCTCCCGTAGGAGT |
b002_16S_A-CF610 | Alphaproteobacteria 16S | Cal Fluor Red 610 (CF610) | TGTACCGACCCTTAACGTTC |
Orsay1 Red | Orsay virus RNA1 | Cal Fluor Red 610 (CF610) | GACATATGTGATGCCGAGAC |
Orsay2 Red | Orsay virus RNA2 | Cal Fluor Red 610 (CF610) | GTAGTGTCATTGTAGGCAGC |
MicroA-CF610 | Nematocida parisii 18S | Cal Fluor Red 610 (CF610) | CTCTGTCCATCCTCGGCAA |
MicroB-CF610 | Nematocida parisii 18S | Cal Fluor Red 610 (CF610) | CTCTCGGCACTCCTTCCTG |
MicroF-CF610 | Nematocida parisii 18S | Cal Fluor Red 610 (CF610) | AGACAAATCAGTCCACGAATT |
MicroSp1A-FAM | Nematocida ausubeli 18S | 5' 6-fluorescein (FAM) | CAGGTCACCCCACGTGCT |
Table 1: List of FISH probe sequences. All FISH probes were commercially purchased with the fluorophore attached to the 5' end (via custom oligonucleotide synthesis; see Table of Materials) and the oligonucleotides were purified by reverse-phase HPLC.
Wild C. elegans are naturally associated with a variety of microbes. Researchers can use RNA FISH to detect and identify these microbes as well as gain insight into their localization in the context of a whole animal. Microbes with desirable or interesting phenotypes can be identified through this method and then isolated for further characterization and sequencing. The abundance of numerous bacterial isolates from wild C. elegans may also be quantified via RNA FISH29. By using the protocol described here, it is also possible to observe known microorganisms inside their hosts and learn more about their interactions. Importantly, Orsay virus and microsporidia are obligate parasites and cannot be cultured independently of the host, so FISH is the standard visualization tool. 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 tipulae19,23.
The main advantage of the FISH protocol is that it offers a simple, quick, and robust method to stain microbes associated with C. elegans. Images produced from FISH staining have a high signal-to-noise ratio, which is achieved by utilizing FISH probes that target the abundant RNA of the SSU within the sample. Because there are typically 30x or higher levels of rRNA than rDNA, most of the signal from FISH staining with probes that target rRNA is due to rRNA rather than rDNA30. Furthermore, RNA FISH makes it possible to see infection or colonization within the context of the whole animal. This visualization is facilitated through co-staining host nuclei with DAPI and/or using fluorescent-marked strains of C. elegans to better highlight the localization of infection or colonization within the sample. For example, microsporidian-specific FISH was used to determine the tissue tropism of Nematocida displodere by using a panel of C. elegans strains with GFP expression in different tissues20. Additionally, this protocol is amenable to changes that allow for researchers to determine the ideal conditions suitable for their specific needs (e.g., adjusting the fixation period, increasing the hybridization temperature).
One critical step in the FISH protocol is fixing the samples. The incubation period following the addition of the fixative is necessary to allow time for the agent to permeabilize the sample. Longer incubation times are not ideal for samples containing transgenic fluorescent proteins due to protein degradation by PFA over time. For samples containing GFP, it is imperative to determine the optimal fixation time to allow for permeabilization, while still maintaining the GFP signal.
FISH can be used to stain for bacteria, viruses, or microsporidia in C. elegans. However, the best type of fixative agent used for FISH depends on the sample and downstream requirements. This protocol presents a PFA solution as the primary fixative agent to stain bacteria and viruses. However, PFA is not sufficient for the visualization of microsporidian spores as it cannot penetrate the spore wall. For visualization of spores, acetone should be used instead. Although, PFA fixation is efficient for FISH labeling of other life stages of microsporidia, including sporoplasms, meronts, and sporonts. Other major differences are seen between acetone fixation and PFA fixation; acetone is more convenient because samples can be quickly stored in the freezer after adding, without the need for washing. However, acetone quickly kills any existing GFP in a transgenic host. PFA is the preferred fixative if it is important to preserve some physiological structures in the host, as acetone-fixed animals appear to be more degraded, making identification of some tissues more difficult. Because the samples are fixed, this FISH protocol does not allow for live imaging of host-microbe interactions in vivo. However, a pulse-chase infection time course followed by FISH staining of samples at various time points can allow one to see some dynamics of microbial infection19,20,31.
Another critical step throughout the protocol is the thorough washing of the samples before and after hybridization. Before hybridization, when collecting the worms into the microfuge tubes, excess bacteria, or other microbes from the NGM plates can be carried with the worm sample. Three washes with PBS-T are standard; however, more washes may be necessary to sufficiently eliminate external microorganisms, especially when using heavily contaminated, wild-isolated C. elegans. When viewing the mounted samples after FISH, there may be some residual FISH probe that produces large amounts of signal in the background of the sample. The wash temperature and the number of washes are important to remove the excess and non-specifically bound probe. To reduce the background fluorescence, it is possible to perform two or three washes with 1 mL of WB every 30 min, instead of one wash with 1 mL of WB for an hour. Different FISH probes may require different wash temperatures. Typically, the wash temperature is 2 °C above the hybridization temperature, but this can be increased if there is too much background fluorescence (high noise).
The FISH protocol utilizes fluorescent probes designed to target species-specific microbial RNA, but FISH probes can be designed for other high-copy transcripts. Other FISH probes may have different melting temperatures, so incubation steps may need to be performed at a higher or lower temperature than described. FISH staining can identify the spatial distribution of microbial colonization or infection within the host, allowing for the characterization of host-microbe and microbe-microbe interactions. One limitation is that only a few conventional fluorophores can be used simultaneously, which reduces the number of different microorganisms that can be detected via FISH at the same time. This limits its use for complex microbiome studies in C. elegans. However, multicolor rRNA-targeted FISH utilizes probes labeled with non-canonical fluorophores that can increase the number of distinct microbial group labels15. Another limitation is that it is difficult to distinguish between closely related species, especially bacteria, that have SSU sequences that are highly similar. However, the extreme sequence divergence between microsporidia species helps to facilitate their differentiation with this protocol (Figure 3)32,33.
Overall, this FISH protocol describes a technique to detect microorganisms within C. elegans. It allows researchers to use a transparent and genetically tractable model system to detect and quantify colonization and infection within the context of an intact animal, as well as identify unique microbial behavior or morphology within the host. A preprint version of this manuscript was posted during review34.
The authors have nothing to disclose.
Thank you to Dr. Marie-Anne Félix for providing us with wild nematode strains. This work was supported by NSF under CAREER Grant 2143718 and California State University under a CSUPERB New Investigator Award to RJL, NIH under R01 AG052622 and R01 GM114139 to ERT, and by an American Heart Association Fellowship to VL.
10% SDS | Invitrogen | AM9822 | |
Acetone | Fisher Scientific | A-11-1 | |
Antifade mounting serum with DAPI (Vectashield) | Vectalab | NC9524612 | |
EDTA | Fisher Scientific | S311-500 | |
FISH probes (see Table 1) | LGC Biosearch Technologies | FISH probes were commercially purchased via custom oligonucleotide synthesis | |
KCl | Fisher Scientific | P217 | |
KH2PO4 | Fisher Scientific | P-286 | |
Na2HPO4 | Fisher Scientific | S375-500 | |
NaCl | Fisher Scientific | S-671 | |
NH4Cl | Fisher Scientific | A-661 | |
Paraformaldehyde | Electron Microscopy Science | 50-980-487 | CAUTION: PFA is a carcinogen. Handle appropriately |
Thermal mixer | Eppendorf | 5384000020 | |
Tris base | Fisher Scientific | BP152 | |
Triton X-100 | Fisher Scientific | BP-151 | |
Tween-20 | Fisher Scientific | BP337-500 |