These protocols enable the study of inherited immunity to microsporidia. Techniques explained here can help us understand how immunity is transmitted across generations and the molecules determining immunity to microsporidia in offspring. C.elegans is a simple and genetically tractable model with a short generation time for assaying inheritance.
The many tools available for the worm can be used to uncover molecular mechanisms of intergenerational immunity. When infecting P0 animals, use a low spore dose that does not significantly impact the parent's ability to produce progeny. This ensures that bleaching yields enough F1 animals for downstream experiments To begin, move the required number of 10-centimeter unseeded nematode growth medium plates from four degrees Celsius to room temperature one day before planned infections.
Just before the infection, pellet down approximately 2, 500 synchronized L1 worms in a microfuge tube at 1, 400 times G for 30 seconds. Remove the supernatant with a pipette tip to leave worms in 50 microliters of M9 media. Add one milliliter of 10X Escherichia coli strain OP50 to the 1-stage worms, and N.parisii spores to the desired concentration.
As an uninfected control, prepare an equivalent tube of L1s and OP50 with a volume of M9 media equivalent to the volume of spore preparation used. Mix the L1 worm sample by vortexing briefly and plate above one milliliter of worms onto a 10-centimeter unseeded nematode growth medium plate. Swirl to ensure the liquid spreads over the whole plate.
Dry the plates in a clean cabinet with the lids off for 10 to 20 minutes or until completely dry before incubating at 21 degrees Celsius for 72 hours. At 72 hours post-infection, examine plates under the dissecting microscope. The plate with infected worms should contain fewer embryos than the plates with uninfected worms.
Then wash the worms off the plates into a microcentrifuge tube using one milliliter M9 media. If many worms remain on the plates, pellet the worms by centrifugation at 1400 times G for 30 seconds. Remove the supernatant and perform additional plate washes.
Centrifuge at 1400 times G for 30 seconds at room temperature to pellet the worms and wash twice with one milliliter of M9 media or until the supernatant is clear. Re-suspend it in a final volume of one milliliter M9 media and mix well by pipetting. Transfer 100 microliters of the suspended worms to a fresh tube and bleach the remaining 900 microliters to break open the adult worms and release F1 embryos for testing.
Perform infections with modifications. as mentioned in the manuscript. At 72 hours post-infection, examine plates under a dissecting microscope.
The plate with naive worms should be smaller and contain less embryos than the plates with primed worms. Then, start the staining and fixing process of worms by washing them off the plates into a microcentrifuge tube using one milliliter of 0.1%Tween 20 in M9 media. If many worms remain on the plates, pellet the worms by centrifugation at 1, 400 times G for 30 seconds at room temperature, remove the supernatant using a pipette tip, and perform additional plate washes.
After pelleting the worms by centrifuging for 30 seconds at 1, 400 times G at room temperature, wash twice with one milliliters of M9 media containing 0.1%Tween 20 or until the supernatant is clear. Remove the supernatant, add 700 microliters of acetone, and leave the worms to fix at room temperature for 10 minutes. Pellet out these fixed worms for 30 seconds at 10, 000 times G at room temperature and give two washes of one milliliter phosphate buffer saline containing 0.1%Tween 20.
Prepare 50 milliliters of DY96 working solution, wrap the container in foil, and store it in a drawer to prevent exposure to light. Add 500 microliters of DY96 working solution to the worm pellet and rotate for 30 minutes at room temperature in darkness. Centrifuge this reaction for 30 seconds at 10, 000 times G at room temperature to pellet the worms and remove the supernatant.
Then, add 15 microliters of the mounting medium with or without DAPI. Pipette 10 microliters of these stained worms onto a microscope slide and place a cover slip over the top. To analyze the DY96-stained worms mounted on slides, perform imaging using the GFP channel of a fluorescence microscope.
To determine the fitness of the worm populations, use a 5X or 10X objective to assay the gravidity of more than 100 worms per condition. At 72 hours post-infection, the F1 animals were fixed and stained with DY96 to assess microsporidia resistance. Infected parental populations and uninfected controls were fixed at 72 hours post-infection and stained with DY96 to visualize the worm embryos and microsporidia spores.
Infected animals are small, contain many microsporidia spores, and produce fewer embryos than healthy, uninfected controls. Assessment of worm gravidity showed that approximately 95%of uninfected animals produced offspring, compared to the infected animals, which were less than 80%Quantifications revealed that approximately 90%of the microsporidia-treated population were infected, as determined by the number of worms containing DY96-stained spores. Quantifications of these fixed animals revealed that the primed worms contained significantly more embryos than their naive counterparts, indicating greater fitness in the face of infection.
Fiji ImageJ was used to determine the parasite burden of individual naive and immune primed worms, which is the percentage of the body filled with fluorescent N.parisii spores. Quantifications revealed a dramatic reduction in the parasite burden of worms that came from infected parents. Further, the calculations of individual worm size revealed that primed worms had a significant growth advantage over naive animals in the face of N.parisii infection.
Imaging studies revealed that while naive animals typically contained multiple spores and several infected cells, the primed animals had far fewer or no spores and typically no sporoplasms. If unprimed and primed F1 populations have similar numbers of gravid and infected animals, count the number of eggs per animal, and analyze infected tissue with ImageJ to find more subtle differences. While direct yellow 96 stains mature spores, fluorescence in situ hybridization can reveal sporoplasms within cells.
This can show you the number of infected cells in primed and unprimed worms. These techniques have shown that the parental transcriptional response can be instrumental in inducing inherited immunity in offspring. Researchers are now investigating the nature of this immunity in offspring.
