The infection of Caenorhabditis elegans by the microsporidian parasite Nematocida parisii enables the worms to produce offspring that are highly resistant to the same pathogen. This is an example of inherited immunity, a poorly understood epigenetic phenomenon. The present protocol describes the study of inherited immunity in a genetically tractable worm model.
Inherited immunity describes how some animals can pass on the “memory” of a previous infection to their offspring. This can boost pathogen resistance in their progeny and promote survival. While inherited immunity has been reported in many invertebrates, the mechanisms underlying this epigenetic phenomenon are largely unknown. The infection of Caenorhabditis elegans by the natural microsporidian pathogen Nematocida parisii results in the worms producing offspring that are robustly resistant to microsporidia. The present protocol describes the study of intergenerational immunity in the simple and genetically tractable N. parisii –C. elegans infection model. The current article describes methods for infecting C. elegans and generating immune-primed offspring. Methods are also given for assaying resistance to microsporidia infection by staining for microsporidia and visualizing infection by microscopy. In particular, inherited immunity prevents host cell invasion by microsporidia, and fluorescence in situ hybridization (FISH) can be used to quantify invasion events. The relative amount of microsporidia spores produced in the immune-primed offspring can be quantified by staining the spores with a chitin-binding dye. To date, these methods have shed light on the kinetics and pathogen specificity of inherited immunity, as well as the molecular mechanisms underlying it. These techniques, alongside the extensive tools available for C. elegans research, will enable important discoveries in the field of inherited immunity.
Inherited immunity is an epigenetic phenomenon whereby parental exposure to pathogens can enable the production of infection-resistant offspring. This type of immune memory has been shown in many invertebrates that lack adaptive immune systems and can protect against viral, bacterial, and fungal disease1. While inherited immunity has important implications for understanding both health and evolution, the molecular mechanisms underlying this protection are largely unknown. This is partly because many of the animals in which inherited immunity has been described are not established model organisms for research. In contrast, studies in the transparent nematode Caenorhabditis elegans benefit from an extensive genetic and biochemical toolkit2,3, a highly annotated genome4,5, and a short generation time. Indeed, research in C. elegans has enabled fundamental advances in the fields of epigenetics and innate immunity6,7, and it is now an established model for studying immune memory8,9.
Microsporidia are fungal pathogens that infect almost all animals and cause lethal infections in immunocompromised humans10. Infection begins when a microsporidia spore injects or "fires" its cellular contents (sporoplasm) into a host cell using a structure called a polar tube. Intracellular replication of the parasite results in the formation of meronts, which ultimately differentiate into mature spores that can exit the cell11,12. While these parasites are detrimental to both human health and food security, there is much still to learn about their infection biology12. Nematocida parisii is a natural microsporidian parasite that replicates exclusively in the intestinal cells of worms, resulting in reduced fecundity and, ultimately, death. The N. parisii –C. elegans infection model has been used to show: (1) the role of autophagy in pathogen clearance13, (2) how microsporidia can exit infected cells non-lytically14, (3) how pathogens can spread from cell-to-cell by forming syncytia15, (4) the proteins N. parisii use to interface with its host16, and (5) the regulation of the transcriptional intracellular pathogen response (IPR)17,18.
Protocols for the infection of C. elegans are described in the current work and can be used to reveal the unique microsporidia biology and dissect the host's response to infection. The microscopy of fixed worms stained with the chitin-binding dye Direct Yellow 96 (DY96) shows the infection spread of chitin-containing microsporidia spores throughout the intestine. DY96 staining also enables the visualization of chitin-containing worm embryos for the simultaneous assessment of worm gravidity (ability to produce embryos) as a readout of host fitness.
Recent work has revealed that C. elegans infected with N. parisii produce offspring that are robustly resistant to the same infection19. This inherited immunity lasts a single generation and is dose-dependent, as offspring from more heavily infected parents are more resistant to microsporidia. Interestingly, N. parisii-primed offspring are also more resistant to the bacterial gut pathogen Pseudomonas aeruginosa, though they are not protected against the natural pathogen Orsay virus19. The present work also shows that immune-primed offspring limit host cell invasion by microsporidia. The method also describes the collection of immune-primed offspring and how FISH can be used to detect N. parisii RNA in intestinal cells to assay host cell invasion and spore firing20.
Together, these protocols provide a solid foundation for studying microsporidia and inherited immunity in C. elegans. It is hoped that future work in this model system will enable important discoveries in the nascent field of inherited immunity. These techniques are also likely to be starting points for investigating microsporidia-induced inherited immunity in other host organisms.
The present study uses wild-type C. elegans Bristol strain N2 grown at 21 °C.
1. Preparation of media
2. Maintenance of C. elegans
3. Synchronization of C. elegans populations using sodium hypochlorite (bleaching)
NOTE: This step is very time-sensitive, so ensure the centrifuge is available before beginning. Alternative, less rapid bleaching protocols are available in the literature and may be used if preferred. To prevent 6% sodium hypochlorite from losing activity over time, store the reagent in the dark at 4 °C and keep it for up to 1 year.
4. Preparation of N. parisii spores
5. Infection of C. elegans with N. parisii to yield immune-primed offspring
6. Testing inherited immunity to N. parisii in C. elegans
7. DY96 staining of C. elegans to visualize embryos and microsporidia spores
NOTE: DY96 is a green fluorescent chitin-binding dye that stains worm embryos and microsporidia spore walls19,15,25. This allows simultaneous monitoring of the fitness and infection status of the worms.
8. Imaging and analysis of DY96-stained worms to assess worm fitness and infection status
9. FISH assay to assess invasion of C. elegans by microsporidia and spore firing
NOTE: The MicroB FISH probe recognizes a conserved region of microsporidian 18s rRNA and can be used to label intracellular sporoplasms (i.e., invaded host cells) and the genetic material within spores.
In the present study, parental populations of C. elegans (P0) were infected at the L1 stage with a low dose of N. parisii spores. These infection conditions are typically used to obtain high numbers of microsporidia-resistant F1 progeny through bleaching of the parents. Infected parental populations and uninfected controls were fixed at 72 hpi and stained with DY96 to visualize the worm embryos and microsporidia spores (Figure 1A). Infected animals are small, contain many microsporidia spores, and produce fewer embryos than healthy uninfected controls. Assessment of worm gravidity showed that ~95% of uninfected animals produced offspring, compared to less than 80% of the infected animals (Figure 1B). Quantifications revealed that ~90% of the microsporidia-treated population were infected, as determined by the number of worms containing DY96-stained spores (Figure 1C). To obtain immune-primed F1 progeny, it is important to use a dose of microsporidia that is high enough to ensure most parents are infected but low enough to ensure that the population can still produce progeny. A table of the microsporidia doses used to obtain the data in this study is provided (Table 1).
Uninfected and infected parent populations (Figure 1) were treated with sodium hypochlorite at 72 hpi to obtain naïve and immune-primed F1 progenies. F1 animals were exposed to a high dose of N. parisii spores at the L1 stage to test for inherited immunity to microsporidia. At 72 hpi, F1 animals were fixed and stained with DY96 to assess microsporidia resistance (Figure 2A). Quantifications of these fixed animals revealed that the primed worms contained significantly more embryos than their naïve counterparts, indicating greater fitness in the face of infection (Figure 2B). FIJI/ImageJ was used to determine the parasite burden of individual naïve and immune-primed worms (i.e., percentage of the body filled with fluorescent N. parisii spores) (Figure 2C). Quantifications revealed a dramatic reduction in the parasite burden of worms that came from infected parents (Figure 2D). Further, calculations of individual worm size revealed that primed worms had a significant growth advantage over naïve animals in the face of N. parisii infection (Figure 2E). These data demonstrate that N. parisii-infected parents produce offspring with high levels of microsporidia resistance.
Previous work has revealed that inherited immunity to microsporidia reduces invasion of intestinal cells by N. parisii19. To visualize differences in host cell invasion, naïve and immune-primed animals (obtained from uninfected or infected parents, as above) were exposed to a maximal dose of N. parisii at the L1 stage. At 30 dpi, the worms were fixed and co-stained, using a FISH probe to detect N. parisii RNA and DY96 to detect N. parisii spore walls. Imaging revealed that, while naïve animals typically contained multiple spores and several infected cells (sporoplasms), the primed animals had far fewer (or no) spores and typically no sporoplasms (Figure 3).
Figure 1: Direct Yellow 96 (DY96) staining of uninfected and N. parisii-infected C. elegans reveals worm gravidity and infection status. (A–C) N2 C. elegans were not infected or infected with a low dose of N. parisii at the L1 stage. At 72 hpi, the worms were fixed, stained with DY96, and imaged to assess worm gravidity and infection status. (A) Representative images are shown. DY96 enables visualization of worm embryos and microsporidia spores (chitin). An inset image of an infected worm is shown on the right-hand side. Embryos are labeled 'E'; N. parisii infection of the intestine is labeled 'Np'. Scale bar for left and middle images = 500 µm. Scale bar for the right image = 100 µm. (B) Worms carrying one or more embryos were scored as gravid and graphed.(C) Worms carrying any number of N. parisii spores in the intestinal cells were scored as infected and graphed. (B–C) Data pooled from 5 independent experiments using n = 100 worms per condition per experiment. Mean ± SEM is shown. The p values were determined by the unpaired two-tailed Student's t-test. Significance was defined as: *p < 0.05; ***p < 0.001. Please click here to view a larger version of this figure.
Figure 2: Direct Yellow 96 (DY96) staining of naïve and immune-primed C. elegans to determine embryos per worm, microsporidia burden, and worm size. (A) N2 C. elegans were infected or not infected with a low dose of N. parisii at the L1 stage. At 72 hpi, the worms were treated with sodium hypochlorite to release embryos. The resulting naïve and immune-primed offspring were infected with a high dose of N. parisii at the L1 stage. At 72 hpi, the worms were fixed, stained with DY96, and imaged to visualize worm embryos and parasite burden. Representative images are shown. Scale bar = 200 µm.(B) The number of embryos per worm was counted and graphed from (A). (C) A screenshot from FIJI/ImageJ shows naïve worms from panel A that have been thresholded to visualize parasite burden (shown in white) using the Thresholding window in the bottom right. Here, individual worms were outlined using the "Polygon selections" tool highlighted in red and defined as "Region of interests" (ROI) using the ROI window in the top right. The Analyze > Measure > Area function was used to quantify the parasite burden of two example worms, shown to be 25.02% and 13.49%. (D) The parasite burden per worm was determined and graphed from (A). (E) From the above images, individual worm size was calculated from animals outlined as in panel C using the Analyze > Measure > Area function. (B, D, and E) Mean ± SEM is shown. The p values were determined by unpaired two-tailed Student's t-test. Significance was defined as: *p < 0.05. **p < 0.01, ***p < 0.001. Please click here to view a larger version of this figure.
Figure 3: Fluorescence in situ hybridization (FISH) against N. parisii 18S rRNA reveals parasite invasion of C. elegans intestinal cells in naïve but not primed animals. N2 C. elegans were infected or not infected with a low dose of N. parisii at the L1 stage. At 72 hpi, the worms were treated with sodium hypochlorite to release embryos. The resulting naïve and immune-primed offspring were infected with a maximal dose of N. parisii at the L1 stage. At 30 mpi, the worms were fixed, subjected to FISH to detect sporoplasms, stained with DY96 to detect microsporidia spore walls, and imaged. Representative images are shown. Inset images for the naïve worm show sporoplasms (red, asterisks), fired spores (green, arrowheads), and an unfired spore (yellow, arrow). The naïve infected worm shown displays 4 sporoplasms. Scale bar = 20µm. Please click here to view a larger version of this figure.
N. parisii dose | Plate concentration (spores/cm2) | Millions of spores per 6 cm plate | Millions of spores per 10 cm plate |
Low | 35,400 | – | 2.7 |
High | 88,400 | 2.5 | – |
Maximal | 2,12,000 | 6 | – |
Table 1: N. parisii doses employed in the study.
The present protocol describes the study of microsporidia and inherited immunity in a simple and genetically tractable N. parisii –C. elegans infection model.
Spore preparation is an intensive protocol that typically yields enough spores for 6 months of experiments, depending on productivity24. Importantly, infectivity must be determined for each new spore "lot" before using it for the experiments. Due to the variability in infectivity between spore preparations, a single lot must be used consistently for all repeats of an experiment. Individual aliquots should not be thawed and re-frozen, as thawing may cause spores to fire and reduce infectivity. As such, spores must only be removed from the -80 °C freezer immediately prior to infection and maintained on ice while on the bench.
In Steps 5-6, the infection assays were outlined. It is important during infection assays to avoid contamination or starvation of the animals, as this may result in additional intergenerational effects that confound immunity in F1s. An important limitation of the inherited immunity assay is that more infected parents produce fewer F1 offspring. Therefore, it is important to strike a careful balance between the parental generation being sufficiently infected to pass on immunity while being healthy enough to produce offspring for testing. Though the current protocol described infection assays for L1 animals, the protocol can be modified to test the impact of microsporidia infection on differently staged parents and the persistence of immunity in older offspring. The methods can also be modified to test the effects of multiple or distinct stresses (e.g., osmotic stress, heavy metal stress, coinfection with another pathogen) on inherited immunity. While inherited immunity to N. parisii de C. elegans is intergenerational (i.e., lasts a single generation)19, the protocol can be adapted to study further generations to test transgenerational effects. While the protocols provided are specific to N. parisii infection, many other nematode-infecting species of microsporidia exist28. The protocols can be adapted to study inherited immunity to other gut-infecting (e.g., Nematocida ausubeli) and epidermal- and muscle-infecting (e.g., Nematocida displodere) microsporidia29,30. C. elegans are also infected by many other natural and human pathogens (bacterial, fungal, and viral). The methods described here can be used to test inherited immunity to other pathogens in C. elegans and the pathogen-specificity of the inherited immune response to N. parisii. C. elegans is a well-established model organism. However, other members of the Caenorhabditis genus, including the hermaphroditic species C. tropicalis and C. briggsae and the male-female species C. kamaaina are also infected to varying degrees with N. parisii31. By making small modifications to the protocols provided, inherited immunity can also be tested in these animals.
In Step 8, quick and simple methods were given for determining differences in microsporidia susceptibility in DY96-stained worms (i.e., % animals infected, % animals gravid). However, sometimes these methods are not sensitive enough to detect small changes in immunity and fitness. This is because a worm with one embryo and many infected cells would be treated the same as a worm with 10 embryos and one infected cell. As such, methods with finer resolution have also been provided for determining infection outcomes in these animals (i.e., % parasite burden, number of embryos, worm size). While both methods can be used, phenotypic differences are typically more pronounced with the latter, making it easier to detect significant differences. Future adaptations to this method may include using machine learning to automate analysis.
In Step 9, techniques were provided for assaying host cell invasion by microsporidia and spore firing using FISH. While DY96 marks microsporidia with a strong fluorescent green signal, other dyes, including the lipophilic stain Nile Red and the chitin-binding Calcofluor White, can also be used to stain N. parisii30,32,33. The present protocol describes fixation using acetone and works well for detecting microsporidia with DY96 and FISH staining. However, fixation with 4% paraformaldehyde can help preserve tissue morphology and improve the signal in some imaging protocols.
Microsporidia is an understudied pathogen, and much of the cell biology of infection remains unknown. C. elegans is a simple model organism, and these protocols can serve as a platform to understand the key processes involved in infection and host defense against this parasite. Inherited immunity is an emerging field, and many questions remain outstanding1. How do infected parents transmit immunity to offspring (maternal provisioning or altered transcriptional regulation in the progeny)? What mediates immunity in offspring (antimicrobial peptides or other immune proteins)? How pathogen-specific are inherited immune responses, and how conserved is inherited immunity between species? Given the extensive tools available with C. elegans, studies building on the protocol described here are well poised to answer these fundamental questions of inherited immunity.
The authors have nothing to disclose.
We are grateful to Winnie Zhao and Yin Chen Wan for providing helpful comments on the manuscript. This work was supported by the Natural Sciences and Engineering Research Council of Canada (Grant #522691522691).
2.0 mm zirconia beads | Biospec Products Inc. | 11079124ZX | |
10 mL syringe | Fisher Scientific | 1482613 | |
5 μm filter | Millipore Sigma | SLSV025LS | |
Axio Imager 2 | Zeiss | – | Fluorescent microscope for imaging of DY96- and FISH- stained worms on microscope slides |
Axio Zoom V.16 Fluorescence Stereo Zoom Microscope | Zeiss | – | For live imaging of fluorescent transgenic animals to visualize the IPR |
Baked EdgeGARD Horizontal Flow Clean Bench | Baker | – | |
Bead disruptor, Genie SI-D238 Analog Disruptor Genie Cell Disruptor, 120 V | Global Industrial | T9FB893150 | |
Cell-VU slide, Millennium Sciences Disposable Sperm Count Cytometers | Fisher Scientific | DRM600 | |
Direct Yellow 96 | Sigma-Aldrich | S472409-1G | |
EverBrite Mounting Medium with DAPI | Biotium | 23001 | |
EverBrite Mounting Medium without DAPI | Biotium | 23002 | |
Fiji/ImageJ software | ImageJ | https://imagej.net/software/fiji/downloads | |
Mechanical rotor | Thermo Sceintific | 415110 / 1834090806873 | Used to spin tubes of bleached embryos for overnight hatching |
MicroB FISH probe | Biosearch Technologies Inc. | – | Synthesized with a Quasar 570 (Cy3) 5' modification and HPLC purified, CTCTCGGCACTCCTTCCTG |
N2 | Wild-type, Bristol strain | Default strain | Caenorhabditis Genetics Center (CGC) |
Sodium dodecyl sulfate (SDS) | Sigma-Aldrich | L3771-100G | |
Sodium hydroxide solution (5 N) | Fisher Chemical | FLSS256500 | |
Sodium hypochlorite solution (6%) | Fisher Chemical | SS290-1 | |
Stemi 508 Stereo Microscope | Zeiss | – | For daily maintenance of worms and counting of L1 worms for assay set ups |
Tween-20 | Sigma-Aldrich | P1379-100ML | |
Vectashield + A16 | Biolynx | VECTH1500 |